Antibody drug conjugates

ABSTRACT

The present disclosure provides antibody drug conjugates comprising STING modulators. Also provided are compositions comprising the antibody drug conjugates. The compounds and compositions are useful for stimulating an immune response in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.63/111,478, filed Nov. 9, 2020; U.S. Provisional Application No.63/232,935, filed Aug. 13, 2021; and U.S. Provisional Application No.63/250,358, filed Sep. 30, 2021, all of which are incorporated herein byreference in their entireties.

FIELD

The present disclosure provides antibody drug conjugates comprisingSTING modulators. Also provided are compositions comprising the antibodydrug conjugates. The compounds and compositions are useful forstimulating an immune response in a subject in need thereof.

BACKGROUND

Antibody drug conjugates (ADCs), a rapidly growing class of targetedtherapeutics, represent a promising new approach toward improving theselectivity and cytotoxic activity of drugs. These therapeutic agentsare comprised of an antibody (or antibody fragment) that can be linkedto a payload drug to form an immunoconjugate. The antibody directs theADC to bind to the targeted cell. The ADC can then be internalized andrelease its payload which provides treatment for the cell. As the ADC isdirected to its targeted cell, the side effects of conjugated drugs maybe lower than those encountered when systematically administering thesame agent.

The adaptor protein STING (Stimulator of Interferon Genes) has beenshown to play a role in the innate immune system. Activation of theSTING pathway triggers an immune response that results in generation ofspecific killer T-cells that shrink tumors and can provide long-lastingimmunity so the tumors do not recur. The activated STING pathway alsocontributes to the antiviral response by producing antiviral andpro-inflammatory cytokines that fight the virus and mobilize both theinnate and adaptive immune systems, ultimately resulting in long-lastingimmunity against the pathogenic virus. The potential therapeuticbenefits of enhancing both innate and adaptive immune responses makeSTING an attractive target for drug discovery. Cyclic dinucleotides mayfunction as STING agonists and are being tested in clinical trials.However, their anionic properties make them poorly membrane permeable,which may limit their ability to engage STING inside the cell, oftenresulting in unwanted distribution of these compounds within thebloodstream.

There is still a need for new STING agonists as well as improved methodsfor delivering them to the targeted cell.

SUMMARY

In a first aspect, the present disclosure provides a compound of formula(I):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

Ab is an anti-CCR2 antibody, anti-CCR2 antibody fragment, or ananti-CCR2 antigen-binding fragment;

D is a modulator of STING activity comprising an amino group on aguanine base, a guanine base derivative, an adenine base, or an adeninebase derivative; and

L is a linker that, is covalently bonded to Ab; and is also covalentlybonded to said amino group on D.

In a first embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein D-L is represented by the formula (Ia):

denotes the point of attachment to Ab;

b is an integer from 1 to 20;

m is 0, 1, 2, 3, or 4;

n is 0 or 1;

each R¹ is independently selected from C₁-C₄alkyl, O—C₁-C₄alkyl, andhalogen;

R² is selected from C₁-C₄alkyl and —(CH₂CH₂O)_(s)—CH₃; wherein s is aninteger from 1 to 10;

R³ and R^(3′) are each independently selected from hydrogen andC₁-C₃alkyl; and

L¹ is a cleavable linker fragment.

In a second embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein D-L is represented by the formula (Ia), wherein:

a is an integer from 1 to 8;

b is an integer from 1 to 10; and

m is 0.

In a third embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein D-L is represented by the formula (Ia), wherein:

m is 0;

n is 0; and

R³ and R^(3′) are each hydrogen.

In a third embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein D-L is represented by the formula (Ia), wherein L¹is

wherein:

is the point of attachment to the nitrogen atom of formula (Ia);

is the point of attachment to Ab;

t is an integer from 1 and 10;

W is absent or a self-immolative group;

Z is absent or a peptide of 2 to 5 amino acids;

U and U′ are independently absent or a spacer; and

Q is a heterobifunctional group;

provided that W and Z are not both absent.

In a fourth embodiment of the first aspect, W is a self-immolative groupselected from

wherein:

is the point of attachment to the carbonyl group; and

is the point of attachment to Z.

In a fifth embodiment of the first aspect, W is

In a sixth embodiment of the first aspect, W is

In a seventh embodiment of the first aspect, Z is a peptide capable ofbeing enzymatically cleaved.

In an eighth embodiment of the first aspect, Z is cathepsin cleavable.

In a ninth embodiment of the first aspect, Z is a two-amino acid peptideselected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe.

In a tenth embodiment of the first aspect, Z is Ala-Val or Val-Ala.

In an eleventh embodiment of the first aspect, U′ is absent and U isselected from

wherein:

is the point of attachment to Z;

is the point of attachment to Q;

p is an integer from 1 to 6;

q is an integer from 1 to 20;

X is O or —CH₂—; and

each r is independently 0 or 1.

In a twelfth embodiment of the first aspect, U′ is absent and U is:

In a thirteenth embodiment of the first aspect, Q is aheterobifunctional group which is attached to U′ or, when U′ is absent,is attached to Ab through chemical or enzyme-mediated conjugation.

In a fourteenth embodiment of the first aspect, Q is selected from

wherein

is the point of attachment to U or, when U is absent, the point ofattachment to Z; and

is the point of attachment to U′, or, when U′ is absent, the point ofattachment to Ab.

In a fifteenth embodiment of the first aspect, Q is:

In a sixteenth embodiment of the first aspect, t is 1.

In a seventeenth embodiment of the first aspect, R² is —CH₃, and R³ andR^(3′) are each hydrogen.

In an eighteenth embodiment of the first aspect, a is from 2 to 6.

In a nineteenth embodiment of the first aspect, b is 1.

In a twentieth embodiment of the first aspect, the amino-substitutedcompound that modulates STING activity is a compound of formula (II):

wherein:

X¹⁰ is SH or OH;

X²⁰ is SH or OH;

Y^(a) is O, S, or CH₂;

Y^(b) is O, S, NH, or NR^(a), wherein R^(a) is C₁-C₄alkyl;

R¹⁰ is hydrogen, fluoro, OH, NH₂, OR^(b), or NHR^(b);

R²⁰ is hydrogen or fluoro;

R³⁰ is hydrogen; R⁴⁰ is hydrogen, fluoro, OH, NH₂, OR^(b), or NHR^(b);or R³⁰ and R⁴⁰ are taken together to form CH₂O;

R⁵⁰ is hydrogen or fluoro;

R^(b) is C₁-C₆alkyl, halo(C₁-C₆)alkyl, or C₃-C₆cycloalkyl;

Ring A¹⁰ is an optionally substituted 5- or 6-membered monocyclicheteroaryl ring containing 1-4 heteroatoms selected from N, O, or S, oran optionally substituted 9 or 10 membered bicyclic heteroaryl ringcontaining 1-5 heteroatoms selected from N, O, or S; wherein ring A¹⁰comprises at least one N atom in the ring, and wherein Y^(b) is attachedto a carbon atom of ring A¹⁰; and

Ring B¹⁰ is an optionally substituted 9 or 10-membered bicyclicheteroaryl ring containing from 2 to 5 heteroatoms selected from N, O,or S; wherein ring B¹⁰ comprises at least two N atoms in the ring;

provided that either ring A¹⁰ or ring B¹⁰ is attached to ‘L’ in formula(I) through the amino group.

In a twenty-first embodiment of the first aspect, the amino-substitutedcompound that modulates STING activity is

wherein

is the point of attachment to ‘L’ in formula (I).

In a twenty-second embodiment of the first aspect, the amino-substitutedcompound that modulates STING activity is a compound of formula (III):

or a pharmaceutically acceptable salt thereof; wherein

X¹⁰ is SH or OH;

X²⁰ is SH or OH;

Y^(c) is O, S, or CH₂;

Y^(d) is O, S, or CH₂;

B¹⁰⁰ is a group represented by formula (B¹-A) or formula (B¹-B):

R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently a hydrogen atom or asubstituent;

R¹⁰⁰⁰ is hydrogen or a bond to the carbonyl group of formula (I);

Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵ and Y¹⁶ are each independently N or CR^(1a),wherein R^(1a) is hydrogen or a substituent;

Z¹¹, Z¹², Z¹³, Z¹⁴, Z¹⁵ and Z¹⁶ are each independently N or C;

R¹⁰⁵ is a hydrogen atom or a substituent;

B²⁰⁰ is a group represented by formula (B²-A) or formula (B²-B):

R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are each independently a hydrogen atom or asubstituent;

R^(100′) is hydrogen or a bond to the carbonyl group of formula (I);

Y²¹, Y²², Y²³, Y²⁴, Y²⁵ and Y²⁶ are each independently N or CR^(2a),wherein R^(2a) is hydrogen or a substituent;

Z²¹, Z²², Z²³, Z²⁴, Z²⁵ and Z²⁶ are each independently N or C; and

R²⁰⁵ is a hydrogen atom or a substituent; wherein R¹⁰⁵ and R²⁰⁵ are eachindependently attached to 2- or 3-position of the 5-membered ring theyare attached to respectively; provided that:

one of B¹⁰⁰ or B²⁰⁰ is attached to ‘L’ in formula (I) through the aminogroup.

In a twenty-third embodiment of the first aspect, the amino-substitutedcompound that modulates STING activity is a compound of formula (IIIa):

or a pharmaceutically acceptable salt thereof; wherein

B¹⁰⁰ is a group represented by formula (B¹-A) or formula (B¹-B):

R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently a hydrogen atom or asubstituent;

R¹⁰⁰⁰ is hydrogen or a bond to the carbonyl group of formula (I);

Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵ and Y¹⁶ are each independently N or CR^(1a),wherein R^(1a) is hydrogen or a substituent;

Z¹¹, Z¹², Z¹³, Z¹⁴, Z¹⁵ and Z¹⁶ are each independently N or C;

R¹⁰⁵ is a hydrogen atom or a substituent;

B²⁰⁰ is a group represented by formula (B²-A) or formula (B²-B):

R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are each independently a hydrogen atom or asubstituent;

R^(100′) is hydrogen or a bond to the carbonyl group of formula (I);

Y²¹, Y²², Y²³, Y²⁴, Y²⁵ and Y²⁶ are each independently N or CR^(2a),wherein R^(2a) is hydrogen or a substituent;

Z²¹, Z²², Z²³, Z²⁴, Z²⁵ and Z²⁶ are each independently N or C; and

R²⁰⁵ is a hydrogen atom or a substituent; wherein R¹⁰⁵ and R²⁰⁵ are eachindependently attached to 2- or 3-position of the 5-membered ring theyare attached to respectively; provided that:

one of B¹⁰⁰ or B²⁰⁰ is:

wherein:

R¹⁸ is hydrogen or C₁₋₆ alkyl; and

R¹⁹ is a halogen atom;

and the other is attached to the ‘L’ group in formula (I) through an—NH— group.

In a twenty-fourth embodiment of the first aspect, the amino-substitutedcompound that modulates STING activity is a compound of formula offormula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are each independently a hydroxy group or a halogen atom;

B¹ is:

R¹⁸ is hydrogen or C₁₋₆ alkyl;

R¹⁹ is a halogen atom;

B² is:

and

Q² and Q⁴ are each independently an oxygen atom or a sulfur atom.

In a twenty-fifth embodiment of the first aspect, the amino-substitutedcompound that modulates STING activity is,

or a pharmaceutically acceptable salt thereof, wherein

is the point of attachment to L.

In a twenty-sixth embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, having the structure of formula (VI):

wherein a is an integer from 1 to 6.

In a twenty-seventh embodiment of the first aspect, Ab is an antibody orfragment thereof that binds human CCR2 or a portion thereof, and iscapable of blocking binding of a chemokine to CCR2 and inhibiting afunction of CCR2.

In a twenty-eighth embodiment of the first aspect, the antibody isselected from the group consisting of monoclonal antibody 1D9 or anantibody which can compete with 1D9 for binding to human CCR2 or aportion of CCR2; MC-21; STI-B020X; UniTI-101; and 4.40A68G.

In a twenty-ninth embodiment of the first aspect, the antibody ismonoclonal antibody 1D9 or an antibody which can compete with 1D9 forbinding to human CCR2 or a portion of CCR2.

In a thirtieth embodiment of the first aspect, the antibody is achimeric antibody, a humanized antibody, a human antibody, a mouseantibody, a rat antibody, a goat antibody, or a rabbit antibody.

In a thirty-first embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment comprises a light chain CDR1 comprising amino acids 24-39 ofSEQ ID NO: 1; a light chain CDR2 comprising amino acids 55-61 of SEQ IDNO: 1; a light chain CDR3 comprising amino acids 94-102 of SEQ ID NO: 1;a heavy chain CDR1 comprising amino acids 31-35 of SEQ ID NO:2; a heavychain CDR2 comprising amino acids 50-68 of SEQ ID NO:2; and a heavychain CDR3 comprising amino acids 101-106 of SEQ ID NO:2.

In a thirty-second embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 2.

In a thirty-third embodiment of the first aspect, the antibody, theanti-CCR2 antibody, anti-CCR2 antibody fragment, or anti-CCR2antigen-binding fragment comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 1.

In a thirty-fourth embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment comprises a heavy chain variable region and a light chainvariable region, wherein the heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 2.

In a thirty-fifth embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment comprises a heavy chain variable region and a light chainvariable region, wherein the light chain variable region comprises theamino acid sequence of SEQ ID NO: 1.

In a thirty-sixth embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 2 and a light chain variable region, whereinthe light chain variable region comprises the amino acid sequence of SEQID NO: 1.

In a thirty-seventh embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment further comprises a heavy chain constant region selected fromhuman immunoglobulins IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂ heavy chainconstant regions.

In a thirty-eighth embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment further comprises a light chain constant region selected fromthe group consisting of human immunoglobulins IgGκ and IgGλ light chainconstant regions.

In a thirty-ninth embodiment of the first aspect, the anti-CCR2antibody, anti-CCR2 antibody fragment, or anti-CCR2 antigen-bindingfragment binds to the same epitope as an antibody comprising a variableheavy chain region of SEQ ID NO: 2 and a variable light chain region ofSEQ ID NO: 1.

In a fortieth embodiment of the first aspect, the anti-CCR2 antibodycomprises a heavy chain region of SEQ ID NO: 3.

In a forty-first embodiment of the first aspect, the anti-CCR2 antibodycomprises a light chain region of SEQ ID NO: 4.

In a forty-second embodiment of the first aspect, the anti-CCR2 antibodycomprises a heavy chain region of SEQ ID NO: 3 and a light chain regionof SEQ ID NO: 4.

In a second aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptablecarriers.

In a first embodiment of the second aspect, the pharmaceuticalcomposition comprises a compound of formula (I) and an antibody thatbinds Programmed Death 1 (PD-1, CD279, hSLE1 or SLEB2).

In a second embodiment of the second aspect, the pharmaceuticalcomposition comprises a compound of formula (I) and an antibody thatbinds Programmed Death Ligand 1 (PD-L1, CD274, or B7H1).

In a third aspect, the present disclosure provides a method of treatingcancer in a subject in need thereof, the method comprising administeringto the subject a pharmaceutically acceptable amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof.

In a first embodiment of the third aspect, the method of treating cancercomprises administering to the subject a pharmaceutically acceptableamount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, and an anti-PD-1 antibody.

In a second embodiment of the third aspect, the method of treatingcancer comprises administering to the subject a pharmaceuticallyacceptable amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and an anti-PD-L1 antibody.

In a third embodiment of the third aspect, the compound of formula (I),or a pharmaceutically acceptable salt thereof, and the anti-PD-1antibody are administered simultaneously.

In a fourth embodiment of the third aspect, the compound of formula (I),or a pharmaceutically acceptable salt thereof, and the anti-PD-1antibody are administered sequentially.

In a fifth embodiment of the third aspect, the compound of formula (I),or a pharmaceutically acceptable salt thereof, and the anti-PD-L1antibody are administered simultaneously.

In a sixth embodiment of the third aspect, the compound of formula (I),or a pharmaceutically acceptable salt thereof, and the anti-PD-L1antibody are administered sequentially.

In a seventh embodiment of the third aspect, the method furthercomprises administering radiation to the subject. In an eighthembodiment of the third aspect, the radiation is particle radiation. Ina ninth embodiment of the third aspect, the radiation is administered byexternal beam radiation.

In a fourth aspect, the present disclosure provides a method forstimulating an immune response in a subject in need thereof, the methodcomprising administering to the subject a pharmaceutically acceptableamount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the preparation of Ab-STING agonist conjugates viastochastic cysteine conjugation.

FIG. 2 depicts the preparation of Ab-STING agonist conjugates viatransglutaminase conjugation.

FIG. 3 depicts the preparation of Ab-STING agonist conjugates viatransglutaminase conjugation.

FIG. 4 depicts the mouse PK profile of Antibody Drug Conjugate B-14.

FIG. 5 depicts the mouse PK profile of Antibody Drug Conjugate B-15.

FIG. 6 depicts the mouse PK profile of Antibody Drug Conjugate B-16.

FIG. 7 depicts the mouse PK profile of Antibody Drug Conjugate B-17.

FIG. 8 depicts the mouse PK profile of Antibody Drug Conjugate B-18.

FIG. 9 depicts the change in body weight over time of mice dosed withADC B-17.

FIG. 10 depicts the change in body weight overtime of mice dosed withADC B-20.

FIG. 11 depicts the antitumor activity of Antibody Drug Conjugate B-21compared to the antitumor activity of its payload alone.

FIG. 12 depicts the change in CCR2 and CD80 expression in monocytes andMDSCs in non-human primates after dosing with Antibody Drug ConjugateB-17.

FIG. 13 depicts the change in serum IL-1RA, IL-6, TNF-α, and IFN-γ innon-human primates after dosing with Antibody Drug Conjugate B-17.

FIG. 14 depicts the non-human primate PK profile of Antibody DrugConjugate B-17.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this disclosure belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context dictates otherwise.

As used herein, the term “or” is a logical disjunction (i.e., and/or)and does not indicate an exclusive disjunction unless expresslyindicated such as with the terms “either,” “unless,” “alternatively,”and words of similar effect.

As used herein, the term “about” refers to ±10%.

Antibody Drug Conjugates

In some embodiments, the present disclosure provides a compound offormula (I),

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

Ab is an anti-CCR2 antibody, anti-CCR2 antibody fragment, or ananti-CCR2 antigen-binding fragment;

D is a modulator of STING activity comprising an amino group on aguanine base, a guanine base derivative, an adenine base, or an adeninebase derivative; and

L is a linker that, is covalently bonded to Ab; and is also covalentlybonded to said amino group on D.

STING Modulator Moiety

The present disclosure provides compounds comprising modulators of STINGactivity. In certain embodiments, the STING modulator is a compound thattargets the STING pathway as an antagonist or an agonist. In someembodiments, the STING modulator is an agonist. In certain embodiments,the STING modulator comprises an amino group on a guanine base, aguanine base derivative, an adenine base, or an adenine base derivative.In some embodiments, the STING modulator is a cyclic dinucleotide, or acyclic dinucleotide-like compound (each, a CDN).

In some embodiments, the STING modulator is a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

X¹⁰ is —SH or —OH;

X²⁰ is —SH or —OH;

Y^(a) is —O—, —S—, or —CH₂—;

Y^(b) is —O—, —S—, —NH—, or —NR^(a)—, wherein R^(a) is C₁-C₄alkyl;

R¹⁰ is hydrogen, fluoro, —OH, —NH₂, —OR^(b), or —NHR^(b);

R²⁰ is hydrogen or fluoro;

R³⁰ is hydrogen; R⁴⁰ is hydrogen, fluoro, —OH, —NH₂, —OR^(b), or—NHR^(b); or R³⁰ and R⁴⁰ are taken together to form —CH₂O—;

R⁵⁰ is hydrogen or fluoro;

R^(b) is C₁-C₆alkyl, halo(C₁-C₆)alkyl, or C₃-C₆cycloalkyl;

Ring A¹⁰ is an optionally substituted 5- or 6-membered monocyclicheteroaryl ring containing 1-4 heteroatoms selected from N, O, or S, oran optionally substituted 9- or 10-membered bicyclic heteroaryl ringcontaining 1-5 heteroatoms selected from N, O, or S; wherein ring A¹⁰comprises at least one N atom in the ring, and wherein Y^(b) is attachedto a carbon atom of ring A¹⁰; and

Ring B¹⁰ is an optionally substituted 9- or 10-membered bicyclicheteroaryl ring containing 2-5 heteroatoms selected from N, O, or S;wherein ring B¹⁰ comprises at least two N atoms in the ring;

provided that either ring A¹⁰ or ring B¹⁰ is attached to ‘L’ in formula(I) through an —NH— group.

As described herein, ring A¹⁰ and ring B¹⁰ can contain one or moresubstituents and thus can be optionally substituted. Suitablesubstituents on the unsaturated carbon atom of a heteroaryl groupinclude, and are generally selected from, -halo, —NO₂, —CN, —R⁺,—C(R⁺)═C(R⁺)₂, —C≡C—R⁺, —OR⁺, —SR^(o), —S(O)R^(o), —SO₂R^(o), —SO₃R⁺,—SO₂N(R⁺)₂, —N(R⁺)₂, —NR⁺C(O)R⁺, —NR⁺C(S)R⁺, —NR⁺C(O)N(R⁺)₂,—NR⁺C(S)N(R⁺)₂, —N(R⁺)C(═NR⁺)—N(R⁺)₂, —N(R⁺)C(═NR⁺)—R^(o), —NR⁺CO₂R⁺,—NR⁺SO₂R^(o), —NR⁺SO₂N(R⁺)₂, —O—C(O)R⁺, —O—CO₂R⁺, —OC(O)N(R⁺)₂, —C(O)R⁺,—C(S)R^(o), —CO₂R⁺, —C(O)—C(O)R⁺, —C(O)N(R⁺)₂, —C(S)N(R⁺)₂,—C(O)N(R⁺)—OR⁺, —C(O)N(R⁺)C(═NR⁺)—N(R⁺)₂, —N(R⁺)C(═NR⁺)—N(R⁺)—C(O)R⁺,—C(═NR⁺)—N(R⁺)₂, —C(═NR⁺)—OR⁺, —N(R⁺)—N(R⁺)₂, —C(═NR⁺)—N(R⁺)—OR⁺,—C(R^(o))═N—OR⁺, —P(O)(R⁺)₂, —P(O)(OR⁺)₂, —O—P(O)—OR⁺, and—P(O)(NR⁺)—N(R⁺)₂, wherein R⁺, independently, is hydrogen or anoptionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic, orheterocyclyl group, or two independent occurrences of R⁺ are takentogether with their intervening atom(s) to form an optionallysubstituted 5-7-membered aryl, heteroaryl, cycloaliphatic, orheterocyclyl. In some embodiments, R⁺, independently, is hydrogen, C₁₋₆aliphatic, or C₃₋₆ cycloaliphatic. Each R^(o) is, independently, anoptionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic, orheterocyclyl group.

As detailed above, in some embodiments, two independent occurrences ofR⁺ (or any other variable similarly defined in the specification andclaims herein), are taken together with their intervening atom(s) toform a monocyclic or bicyclic ring selected from 3-13-memberedcycloaliphatic, 3-12-membered heterocyclyl having 1-5 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, 6-10-memberedaryl, or 5-10-membered heteroaryl having 1-5 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

In some embodiments the STING modulator is a compound of formula (IIA):

or a pharmaceutically acceptable salt thereof, wherein R¹⁰ and R⁴⁰ areeach independently hydrogen, fluoro, —OH, or —OCH₂CF₃ and rings A¹⁰ andB¹⁰ are as defined for the compound of formula (II), provided thateither ring A¹⁰ or ring B¹⁰ is attached to ‘L’ through an —NH— group.

In some embodiments, ring A¹⁰ is an optionally substituted 6-memberedmonocyclic heteroaryl ring containing 1, 2, or 3 nitrogen atoms.

In some embodiments, ring B¹⁰ is:

wherein:

Z¹⁰, Z²⁰, Z³⁰, and Z⁴⁰ are each independently N or CR²⁰⁰;

R²¹⁰ is hydrogen or C₁-C₆alkyl, halo(C₁-C₆)alkyl, or C₃-C₆cycloalkyl;

R²³⁰ is hydrogen or —NH₂; and

R²⁰⁰, R²²⁰, and R²⁴⁰ are each independently hydrogen, halogen, —OH,—NH₂, —CN, C₁-C₆alkyl, halo(C₁-C₆)alkyl, or C₃-C₆cycloalkyl.

In some embodiments the STING modulator is:

or a pharmaceutically acceptable salt thereof, wherein

is the point of attachment to the ‘L’ group of the parent molecularmoiety.

In some embodiments, the STING modulator is a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

X¹⁰ is SH or OH;

X²⁰ is SH or OH;

Y^(c) is O, S, or CH₂;

Y^(d) is O, S, or CH₂;

R¹⁰⁵ and R²⁰⁵ are each independently hydrogen or a substituent, whereinR¹⁰⁵ and R²⁰⁵ are each independently attached to 2- or 3-position of the5-membered ring they are attached to respectively;

B¹⁰⁰ is a group represented by formula (B¹-A) or formula (B¹-B):

R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently a hydrogen atom or asubstituent;

R¹⁰⁰⁰ is hydrogen or a bond to the carbonyl group of formula (I);

Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵ and Y¹⁶ are each independently N or CR^(1a);

Z¹¹, Z¹², Z¹³, Z¹⁴, Z¹⁵ and Z¹⁶ are each independently N or C;

R^(1a) is a hydrogen atom or a substituent;

B²⁰⁰ is a group represented by formula (B²-A) or formula (B²-B).

R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are each independently a hydrogen atom or asubstituent;

R^(100′) is hydrogen or a bond to the carbonyl group of formula (I);

Y²¹, Y²², Y²³, Y²⁴, Y²⁵ and Y²⁶ are each independently N or CR^(2a);

Z²¹, Z²², Z²³, Z²⁴, Z²⁵ and Z²⁶ are each independently N or C; and

R^(2a) is a hydrogen atom or a substituent;

provided that one of B¹⁰⁰ or B²⁰⁰ is attached to the carbonyl group offormula (I) through an —NH— group.

As described herein, compounds of formula (III) and formula (IIIa)(below) comprise substituents at certain positions. Suitablesubstituents include a halogen atom, a cyano group, a nitro group, anoptionally substituted hydrocarbon group, an optionally substitutedheterocyclic group, an acyl group, an optionally substituted aminogroup, an optionally substituted carbamoyl group, an optionallysubstituted thiocarbamoyl group, an optionally substituted sulfamoylgroup, an optionally substituted hydroxy group, an optionallysubstituted sulfanyl (SH) group and an optionally substituted silylgroup, wherein the optionally substituted groups have one or moresubstituents selected from substituent group A:

“Substituent group A:”(1) a halogen atom,(2) a nitro group,(3) a cyano group,(4) an oxo group,(5) a hydroxy group,(6) an optionally halogenated C₁₋₆ alkoxy group,(7) a C₆₋₁₄ aryloxy group (e.g., phenoxy, naphthoxy),(8) a C₇₋₁₆ aralkyloxy group (e.g., benzyloxy),(9) a 5- to 14-membered aromatic heterocyclyloxy group (e.g.,pyridyloxy),(10) a 3- to 14-membered non-aromatic heterocyclyloxy group (e.g.,morpholinyloxy, piperidinyloxy),(11) a C₁₋₆ alkyl-carbonyloxy group (e.g., acetoxy, propanoyloxy),(12) a C₆₋₁₄ aryl-carbonyloxy group (e.g., benzoyloxy, 1-naphthoyloxy,2-naphthoyloxy),(13) a C₁₋₆ alkoxy-carbonyloxy group (e.g., methoxycarbonyloxy,ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy),(14) a mono- or di-C₁₋₆ alkyl-carbamoyloxy group (e.g.,methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy,diethylcarbamoyloxy),(15) a C₆₋₁₄ aryl-carbamoyloxy group (e.g., phenylcarbamoyloxy,naphthylcarbamoyloxy),(16) a 5- to 14-membered aromatic heterocyclylcarbonyloxy group (e.g.,nicotinoyloxy),(17) a 3- to 14-membered non-aromatic heterocyclylcarbonyloxy group(e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy),(18) an optionally halogenated C₁₋₆ alkylsulfonyloxy group (e.g.,methylsulfonyloxy, trifluoromethylsulfonyloxy),(19) a C₆₋₁₄ arylsulfonyloxy group optionally substituted by a C₁₋₆alkyl group (e.g., phenylsulfonyloxy, toluenesulfonyloxy),(20) an optionally halogenated C₁₋₆ alkylthio group,(21) a 5- to 14-membered aromatic heterocyclic group,(22) a 3- to 14-membered non-aromatic heterocyclic group,(23) a formyl group,(24) a carboxy group,(25) an optionally halogenated C₁₋₆ alkyl-carbonyl group,(26) a C₆₋₁₄ aryl-carbonyl group,(27) a 5- to 14-membered aromatic heterocyclylcarbonyl group,(28) a 3- to 14-membered non-aromatic heterocyclylcarbonyl group,(29) a C₁₋₆ alkoxy-carbonyl group,(30) a C₆₋₁₄ aryloxy-carbonyl group (e.g., phenyloxycarbonyl,1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl),(31) a C₇₋₁₆ aralkyloxy-carbonyl group (e.g., benzyloxycarbonyl,phenethyloxycarbonyl),(32) a carbamoyl group,(33) a thiocarbamoyl group,(34) a mono- or di-C₁₋₆ alkyl-carbamoyl group,(35) a C₆₋₁₄ aryl-carbamoyl group (e.g., phenylcarbamoyl),(36) a 5- to 14-membered aromatic heterocyclylcarbamoyl group (e.g.,pyridylcarbamoyl, thienylcarbamoyl),(37) a 3- to 14-membered non-aromatic heterocyclylcarbamoyl group (e.g.,morpholinylcarbamoyl, piperidinylcarbamoyl),(38) an optionally halogenated C₁₋₆ alkylsulfonyl group,(39) a C₆₋₁₄ arylsulfonyl group,(40) a 5- to 14-membered aromatic heterocyclylsulfonyl group (e.g.,pyridylsulfonyl, thienylsulfonyl),(41) an optionally halogenated C₁₋₆ alkylsulfinyl group,(42) a C₆₋₁₄ arylsulfinyl group (e.g., phenylsulfinyl,1-naphthylsulfinyl, 2-naphthylsulfinyl),(43) a 5- to 14-membered aromatic heterocyclylsulfinyl group (e.g.,pyridylsulfinyl, thienylsulfinyl),(44) an amino group,(45) a mono- or di-C₁₋₆ alkylamino group (e.g., methylamino, ethylamino,propylamino, isopropylamino, butylamino, dimethylamino, diethylamino,dipropylamino, dibutylamino, N-ethyl-N-methylamino),(46) a mono- or di-C₆₋₁₄ arylamino group (e.g., phenylamino),(47) a 5- to 14-membered aromatic heterocyclylamino group (e.g.,pyridylamino),(48) a C₇₋₁₆ aralkylamino group (e.g., benzylamino),(49) a formylamino group,(50) a C₁₋₆ alkyl-carbonylamino group (e.g., acetylamino,propanoylamino, butanoylamino),(51) a (C₁₋₆ alkyl)(C₁₋₆ alkyl-carbonyl) amino group (e.g.,N-acetyl-N-methylamino),(52) a C₆₋₁₄ aryl-carbonylamino group (e.g., phenylcarbonylamino,naphthylcarbonylamino),(53) a C₁₋₆ alkoxy-carbonylamino group (e.g., methoxycarbonylamino,ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino,tert-butoxycarbonylamino),(54) a C₇₋₁₆ aralkyloxy-carbonylamino group (e.g.,benzyloxycarbonylamino),(55) a C₁₋₆ alkylsulfonylamino group (e.g., methylsulfonylamino,ethylsulfonylamino),(56) a C₆₋₁₄ arylsulfonylamino group optionally substituted by a C₁₋₆alkyl group (e.g., phenylsulfonylamino, toluenesulfonylamino),(57) an optionally halogenated C₁₋₆ alkyl group,(58) a C₂₋₆ alkenyl group,(59) a C₂₋₆ alkynyl group,(60) a C₃₋₁₀ cycloalkyl group,(61) a C₃₋₁₀ cycloalkenyl group, and(62) a C₆₋₁₄ aryl group.

In some embodiments the STING modulator is a compound of formula (IIIa),or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof; whereinB¹⁰⁰ is a group represented by formula (B¹-A) or formula (B¹-B):

R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently a hydrogen atom or asubstituent;R¹⁰⁰⁰ is hydrogen or a bond to the carbonyl group of formula (I);Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵ and Y¹⁶ are each independently N or CR^(1a),wherein R^(1a) is hydrogen or a substituent;Z¹¹, Z¹², Z¹³, Z¹⁴, Z¹⁵ and Z¹⁶ are each independently N or C;R¹⁰⁵ is a hydrogen atom or a substituent;B²⁰⁰ is a group represented by formula (B²-A) or formula (B²-B):

R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are each independently a hydrogen atom or asubstituent;

R^(100′) is hydrogen or a bond to the carbonyl group of formula (I);Y²¹, Y²², Y²³, Y²⁴, Y²⁵ and Y²⁶ are each independently N or CR^(2a),wherein R^(2a) is hydrogen or a substituent;Z²¹, Z²², Z²³, Z²⁴, Z²⁵ and Z²⁶ are each independently N or C; andR²⁰⁵ is a hydrogen atom or a substituent; wherein R¹⁰⁵ and R²⁰⁵ are eachindependently attached to 2- or 3-position of the 5-membered ring theyare attached to respectively; provided that:one of B¹⁰⁰ or B²⁰⁰ is:

wherein:R¹⁸ is hydrogen or C₁₋₆ alkyl; andR¹⁹ is a halogen atom;and the other is attached to the carbonyl group of formula (I) throughan —NH— group.

In some embodiments the STING modulator is a compound of formula (IV),or a pharmaceutically acceptable salt thereof:

or a pharmaceutically acceptable salt thereof, wherein:R¹ and R² are each independently a hydroxy group or a halogen atom; B¹is:

R¹⁸ is hydrogen or C₁₋₆ alkyl;R¹⁹ is a halogen atom;

B² is:

andQ² and Q⁴ are each independently an oxygen atom or a sulfur atom.

In some embodiments, the cyclic dinucleotide is:

or a pharmaceutically acceptable salt thereof, wherein

is the point of ‘L.’.

Linker Moiety

The group “L” is a linker. As used herein, the term “linker” refers toany chemical moiety capable of connecting the antibody, antibodyfragment, or antigen-binding fragment (Ab) to the drug-containing moietywithin the compounds of formula (I) and (IV). The linker can bebranched, and can be substituted with from 1 to 20 drug-containingmoieties. In some embodiments, the linker can be substituted with from 1to 10 drug-containing moieties. In some embodiments, the linker can besubstituted with from 1 to 5 drug-containing moieties. In someembodiments, the linker can be substituted with one or twodrug-containing moieties. In some embodiments, the linker can besubstituted with one drug-containing moiety.

In some embodiments the linker “L” is a cleavable linker. In certainembodiments the linker can be susceptible to acid-induced cleavage,photo-induced cleavage, enzymatic cleavage, or the like, at conditionsunder which the drug and/or antibody can remain active. In someembodiments, the cleavable linker can be cleaved enzymatically. In someembodiments, the cleavable linker can be cleaved by a protease,peptidase, esterase, glycosidase, phosphodiesterase, phosphatase, orlipase. In some embodiments, the cleavable linker can be cleaved by aprotease. Examples of proteases include, but are not limited to,cathepsin B, VAGP tetrapeptide, and the like.

In certain embodiments, the linker can be any of those disclosed in PCTpublications WO 2018/200812, WO 2018/100558, which are incorporated byreference in their entireties.

In certain embodiments, “L” has the formula:

wherein:

is the point of attachment to the nitrogen atom; and

is the point of attachment to Ab.

In some embodiments, “L” has the formula:

wherein:

is the point of attachment to the nitrogen atom;

is the point of attachment to the antibody;

The group “W” is absent or a self-immolative group. As used herein, theterm “self-immolative,” refers to a group that undergoes an electroniccascade which results in the release of the group to which it isattached. In some embodiments, the self-immolative group comprises oneor more groups which can undergo 1,4-elimination, 1,6-elimination,1,8-elimination, 1,6-cyclization elimination, 1,5-cyclizationelimination, 1,3-cyclization elimination, intramolecular 5-exo-trigcyclization, and/or 6-exo-trig cyclization. In certain embodiments theself-immolative group can be any of those disclosed in PCT publicationsWO 2018/200812, WO 2018/100558, which are incorporated by reference intheir entireties.

The group “Z” is absent or a peptide of 2 to 5 amino acids. In certainembodiments, the peptide is the site of cleavage of the linker, therebyfacilitating release of the drug upon exposure to intracellularproteases, such as lysosomal enzymes (Doronina et al. (2003) Nat.Biotechnol. 21:778-784). Examples of peptides having two amino acidsinclude, but are not limited to, alanine-alanine (Ala-Ala),valine-alanine (VA or Val-Ala), valine-citrulline (VC or Val-Cit),alanine-phenylalanine (AF or Ala-Phe); phenylalanine-lysine (FK orPhe-Lys); phenylalanine-homolysine (Phe-Homolys); andN-methyl-valine-citrulline (Me-Val-Cit). Examples of peptides havingthree amino acids include, but are not limited to,glycine-valine-citrulline (Gly-Val-Cit) and glycine-glycine-glycine(Gly-Gly-Gly). The amino acid combinations above can also be present inthe reverse order (i.e., Cit-Val).

The peptides of the present disclosure may comprise naturally-occurringand/or non-natural amino acid residues. The term “naturally-occurringamino acid” refer to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu,Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. “Non-natural aminoacids” (i.e., amino acids do not occur naturally) include, by way ofnon-limiting example, homoserine, homoarginine, citrulline,phenylglycine, taurine, iodotyrosine, seleno-cysteine, norleucine(“Nle”), norvaline (“Nva”), beta-alanine, L- or D-naphthalanine,ornithine (“Orn”), and the like. Peptides can be designed and optimizedfor enzymatic cleavage by a particular enzyme, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Amino acids also include the D-forms of natural and non-natural aminoacids. “D-” designates an amino acid having the “D” (dextrorotary)configuration, as opposed to the configuration in the naturallyoccurring (“L-”) amino acids. Natural and non-natural amino acids can bepurchased commercially (Sigma Chemical Co., Advanced Chemtech) orsynthesized using methods known in the art.

The groups “U” and “U′” are independently absent or a spacer. As usedherein, the term “spacer,” refers to chemical moiety that serves as aconnector. In the present disclosure the spacer can connect theantibody, antibody fragment, or antigen fragment to theheterobifunctional group and/or connect the heterobifunctional group topeptide “Z,” or, when “Z” is absent, to group “W”. Non-limitingexemplary spacers include —NH—, —S—, —O—, —NHC(═O)CH₂CH₂—,—S(═O)₂—CH₂CH₂—, —C(═O)NHNH—, —C(═O)O—, —C(═O)NH—, —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂═CH₂—, —C≡C—, —CH═N—O—, polyethylene glycol (PEG),

In the compounds of the present disclosure, when “U” is present, it canbe a branched group substituted by from 1 to 10 “—C(O)—W—Z—” groups. Insome embodiments, “U” is substituted by from 1 to 5 “—C(O)—W—Z—” groups.In some embodiments, “U” is substituted with 1 or 2 “—C(O)—W—Z—” groups.In some embodiments, “U” is substituted with 1 “—C(O)—W—Z—” group. Incertain embodiments the spacer can be any of those disclosed in PCTpublications WO 2018/200812, WO 2018/100558, which are incorporated byreference in their entireties.

Group “Q” is a heterobifunctional group. In the present disclosure, theterm “heterobifunctional group” refers to a chemical moiety thatconnects the linker of which it is a part to the antibody, antibodyfragment, or antigen-binding fragment. See, e.g., WO 2017/191579.Heterobifunctional groups are characterized as having different reactivegroups at either end of the chemical moiety. The heterobifunctionalgroup may be attached directly to “Ab,” or alternatively, may connectthrough linker “U′”. Attachment to “Ab,” can be accomplished throughchemical or enzymatic conjugation, or a combination of both. Chemicalconjugation involves the controlled reaction of accessible amino acidresidues on the surface of the antibody with a reaction handle on “Q” or“U′”. Examples of chemical conjugation include, but are not limited to,lysine amide coupling, cysteine coupling, and coupling via a non-naturalamino acid incorporated by genetic engineering, wherein non-naturalamino acid residues with a desired reaction handle are installed onto“Ab”. In enzymatic conjugation, an enzyme mediates the coupling of thelinker with an accessible amino residue on the antibody, antibodyfragment, or antigen-binding fragment. Examples of enzymatic conjugationinclude, but are not limited to, transpeptidation using sortase,transpeptidation using microbial transglutaminase, and N-glycanengineering. Chemical conjugation and enzymatic conjugation may also beused sequentially. For example, enzymatic conjugation can also be usedfor installing unique reaction handles on “Ab” to be utilized insubsequent chemical conjugation. In certain embodiments theheterobifunctional group can be any of those disclosed in PCTpublications WO 2018/200812, WO 2018/100558, which are incorporated byreference in their entireties.

In some embodiments, “Q” is selected from

wherein

is the point of attachment to U, or, when U is absent, the point ofattachment to Z; and

is the point of attachment to U′, or, when U′ is absent, the point ofattachment to Ab.

In certain embodiments, the present disclosure provides a compound offormula (XX):

or a pharmaceutically acceptable salt thereof, wherein n, m, a, t,D-NH—, R¹, R², R³, R^(3′), W, Z, and U are as described herein andwherein Q* is reactive functional group capable of conjugating to anantibody, antibody fragment, or antigen-binding fragment. Examples ofsuitable Q* groups include, but are not limited to, activated carboxylicacid groups, such as acid chloride —C(O)—Cl and acid anhydrides,haloacetamide, maleimide, alkyne, cycloalkyne, such as a cyclooctyne,oxanoboradiene, norbornene, azide, diaryl tetrazine, monoaryl tetrazine,aldehyde, ketone, hydroxylamine, vinylsulfone, and aziridine. In certainembodiments the reactive functional group can be any of those disclosedin PCT publications WO 2018/200812, WO 2018/100558, which areincorporated by reference in their entireties.

Anti-CCR2 Antibodies, Antibody Fragments, and Antigen-Binding Fragments

Group “Ab” is an anti-CCR2 antibody, anti-CCR2 antibody fragment, or ananti-CCR2 antigen-binding fragment. An antibody is a protein generatedby the immune system that is capable of recognizing and binding to aspecific antigen. A target antigen generally has numerous binding sites,also called epitopes, recognized by CDRs on multiple antibodies. Eachantibody that specifically binds to a different epitope has a differentstructure. Thus, one antigen may have more than one correspondingantibody. The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, single domain antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. Antibodies may be murine, human, humanized,chimeric, or derived from other species. (Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., GarlandPublishing, New York).

Useful anti-CCR2 antibodies, antibody fragments, and antigen-bindingfragments include an antibody (immunoglobulin) or functional fragmentthereof (e.g., an antigen-binding fragment) which binds to a mammalianCC-chemokine receptor 2 (also referred to as CCR2, CKR-2, CD192, MCP-1RAor MCP-1RB) or portion of the receptor. In one embodiment, the antibodyor fragment thereof has specificity for human or rhesus CCR2 or aportion thereof. In another embodiment, the antibody or fragment blocksbinding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptorand inhibits function associated with binding of the ligand to thereceptor (e.g., leukocyte trafficking). For example, as describedherein, antibodies and fragments thereof useful in the presentdisclosure bind human or rhesus CCR2 or a portion thereof, and can blockbinding of a chemokine (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to thereceptor and inhibit function associated with binding of the chemokineto the receptor. In one embodiment, the antibody is monoclonal antibody(mAb) LS132.1D9 (1D9) or an antibody which can compete with 1D9 forbinding to human CCR2 or a portion of human CCR2. Functional fragmentsof the foregoing antibodies are also envisioned.

In some embodiments, a humanized immunoglobulin or antigen-bindingfragment thereof having binding specificity for CCR2 is employed, saidimmunoglobulin comprising an antigen binding region of nonhuman origin(e.g., rodent) and at least a portion of an immunoglobulin of humanorigin (e.g., a human framework region, a human constant region of thegamma type). In one embodiment, the humanized immunoglobulin or fragmentthereof can compete with 1D9 for binding to CCR2. In one embodiment, theantigen binding region of the humanized immunoglobulin is derived frommonoclonal antibody 1D9 (e.g., an immunoglobulin comprising the variableregions of the light and heavy chains as shown below).

For example, the humanized immunoglobulin or antigen-binding fragmentthereof can comprise an antigen binding region comprising at least onecomplementarity determining region (CDR) of nonhuman origin, and aframework region (FR) derived from a human framework region. In oneaspect, the humanized immunoglobulin having binding specificity for CCR2comprises a light chain comprising at least one CDR derived from anantibody of nonhuman origin which binds CCR2 and a FR derived from alight chain of human origin (e.g., from HF-21/28), and a heavy chaincomprising a CDR derived from an antibody of nonhuman origin which bindsCCR2 and a FR derived from a heavy chain of human origin (e.g., from4B4′CL). In another aspect, the light chain comprises three CDRs derivedfrom the light chain of the 1D9 antibody, and the heavy chain comprisesthree CDRs derived from the heavy chain of the 1D9 antibody.

In one embodiment, the humanized immunoglobulin having bindingspecificity for CCR2 comprises CDR1, CDR2 and CDR3 of the light chain ofthe 1D9 antibody, and a human light chain FR, and comprises CDR1, CDR2and CDR3 of the heavy chain of the 1D9 antibody, and a human heavy chainFR. In one embodiment, the humanized immunoglobulin comprises thehumanized heavy and light chains described herein (e.g., a humanizedlight chain comprising the variable region of the light chain shownbelow, a humanized heavy chain comprising the variable region of theheavy chain shown below. Also encompassed are humanized immunoglobulinscomprising one or more humanized light and/or heavy chains.

The following shows the amino acid sequence of the kappa light chainvariable region (VL) of the humanized 1D9 antibody. The CDRs arehighlighted in bold:

(SEQ ID NO: 1) DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTFLNWFQQRPGQSPR RLIYLVSKLD SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCWQGTHFP YTFGQGTRLE IK. 

The following shows the amino acid sequence of the heavy chain variableregion (VH) of the humanized 1D9 antibody. The CDRs are highlighed inbold:

(SEQ ID NO: 2) EVQLVESGGG LVKPGGSLRL SCAASGFTFS AYAMNWVRQAPGKGLEWVGR IRTKNNNYAT YYADSVKDRF TISRDDSKNTLYLQMNSLKT EDTAVYYCTT FYGNGVWGQG TLVTVSS. 

In certain embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment comprises a light chainCDR1 comprising amino acids 24-39 of SEQ ID NO: 1; a light chain CDR2comprising amino acids 55-61 of SEQ ID NO: 1; a light chain CDR3comprising amino acids 94-102 of SEQ ID NO: 1; a heavy chain CDR1comprising amino acids 31-35 of SEQ ID NO:2; a heavy chain CDR2comprising amino acids 50-68 of SEQ ID NO:2; and a heavy chain CDR3comprising amino acids 101-106 of SEQ ID NO:2.

In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment comprises a heavy chainvariable region and a light chain variable region, wherein the lightchain variable region comprises the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 2 and alight chain variable region, wherein the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment further comprises aheavy chain constant region. In some embodiments, the heavy chainconstant region is selected from human immunoglobulins IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂ heavy chain constant regions.

In some embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment further comprises alight chain constant region. In some embodiments, light chain constantregion is selected from the group consisting of human immunoglobulinsIgGκ and IgGλ light chain constant regions.

In certain embodiments, the anti-CCR2 antibody, anti-CCR2 antibodyfragment, or anti-CCR2 antigen-binding fragment binds to the sameepitope as an antibody comprising a variable heavy chain region of SEQID NO: 2 and a variable light chain region of SEQ ID NO: 1.

“Percent identity” refers to the extent of identity between twosequences (e.g., amino acid sequences or nucleic acid sequences).Percent identity can be determined by aligning two sequences,introducing gaps to maximize identity between the sequences. Alignmentscan be generated using programs known in the art. For purposes herein,alignment of nucleotide sequences can be performed with the blastnprogram set at default parameters, and alignment of amino acid sequencescan be performed with the blastp program set at default parameters (seeNational Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov).

A CCR2 antibody that “binds to the same epitope” as a reference CCR2antibody refers to an antibody that binds to the same CCR2 amino acidresidues as the reference CCR2 antibody. The ability of a CCR2 antibodyto bind to the same epitope as a reference CCR2 antibody is determinedby a hydrogen/deuterium exchange assay (see Coales et al. Rapid Commun.Mass Spectrom. 2009; 23: 639-647).

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human CCR2, comprises the six CDRs of theantibody listed in SEQ ID NO: 1 and SEQ ID NO: 2, and comprises a VHcomprising a sequence at least 80% identical to the VH sequence of SEQID NO: 2 and a VL comprising a sequence at least 80% identical to the VLsequence of SEQ ID NO: 1. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human CCR2,comprises the six CDRs of the antibody listed in SEQ ID NO: 1 and SEQ IDNO: 2 (i.e., the three VH CDRs of the antibody listed in SEQ ID NO: 2and the three VL CDRs of SEQ ID NO: 1), and comprises a VH comprising asequence at least 85% identical to the VH sequence of SEQ ID NO: 2 and aVL comprising a sequence at least 85% identical to the VL sequence ofSEQ ID NO: 1.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human CCR2, comprises the six CDRs of theantibody listed SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VH CDRsof SEQ ID NO: 2 and the three VL CDRs of SEQ ID NO: 1), and comprises aVH comprising a sequence at least 90% identical to the VH sequence ofSEQ ID NO: 2 and a VL comprising a sequence at least 90% identical tothe VL sequence of SEQ ID NO: 1. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human CCR2,comprises the six CDRs of the antibody listed in SEQ ID NO: 1 and SEQ IDNO: 2 (i.e., the three VH CDRs of the antibody and the three VL CDRs ofSEQ ID NO: 1), and comprises a VH comprising a sequence at least 95%identical to the VH sequence of SEQ ID NO: 2 and a VL comprising asequence at least 95% identical to the VL sequence of SEQ ID NO: 1.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human CCR2, comprises the six CDRs of theantibody listed in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VHCDRs of SEQ ID NO: 2 and the three VL CDRs of the SEQ ID NO: 1), andcomprises a VH comprising a sequence at least 96% identical to the VHsequence of SEQ ID NO: 2 and a VL comprising a sequence at least 96%identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, anantibody or antigen-binding fragment thereof described herein binds tohuman CCR1, comprises the six CDRs of the antibody listed in SEQ ID NO:1 and SEQ ID NO: 2 (i.e., the three VH CDRs of SEQ ID NO: 2 and thethree VL CDRs of SEQ ID NO: 1), and comprises a VH comprising a sequenceat least 97% identical to the VH sequence of SEQ ID NO: 2 and a VLcomprising a sequence at least 970% identical to the VL sequence of SEQID NO. In certain embodiments, an antibody or antigen-binding fragmentthereof described herein binds to human CCR2, comprises the six CDRs ofthe antibody listed in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VHCDRs of SEQ ID NO: 2 and the three VL CDRs of SEQ ID NO: 1), andcomprises a VH comprising a sequence at least 98% identical to the VHsequence of SEQ ID NO: 2 and a VL comprising a sequence at least 98%identical to the VL sequence of SEQ ID NO: 1. In certain embodiments, anantibody or antigen-binding fragment thereof described herein binds tohuman CCR2, comprises the six CDRs of the antibody listed in SEQ ID NO:1 and SEQ ID NO: 2 (i.e., the three VH CDRs of SEQ ID NO: 2 the three VLCDRs of SEQ ID NO: 1), and comprises a VH comprising a sequence at least99% identical to the VH sequence of SEQ ID NO: 2 and a VL comprising asequence at least 99% identical to the VL sequence of SEQ ID NO: 1.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human CCR2, comprises the six CDRs of theantibody listed in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VHCDRs of SEQ ID NO: 2 and the three VL CDRs of SEQ ID NO: 1), comprises aVH comprising a sequence at least 80% identical to the VH sequence ofSEQ ID NO: 2 and a VL comprising a sequence at least 80% identical tothe VL sequence of SEQ ID NO: 1, and binds to human, cynomolgus monkey,rat, and/or mouse CCR2. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to CCR2,comprises the six CDRs of the antibody listed in SEQ ID NO: 1 and SEQ IDNO: 2 (i.e., the three VH CDRs of SEQ ID NO: 2 and the three VL CDRs ofSEQ ID NO: 1), comprises a VH comprising a sequence at least 85%identical to the VH sequence of SEQ ID NO: 2 and a VL comprising asequence at least 85% identical to the VL sequence of SEQ ID NO: 1, andbinds to human, cynomolgus monkey, rat, and/or mouse CCR2.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human CCR2, comprises the six CDRs of theantibody listed in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VHCDRs of the antibody and the three VL CDRs of SEQ ID NO: 1), comprises aVH comprising a sequence at least 90% identical to the VH sequence ofSEQ ID NO: 2 and a VL comprising a sequence at least 90% identical tothe VL sequence of SEQ ID NO: 1, and binds to human, cynomolgus monkey,rat, and/or mouse CCR2. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human CCR2,comprises the six CDRs of the antibody listed in SEQ ID NO: 1 and SEQ IDNO: 2 (i.e., the three VH CDRs of SEQ ID NO: 2 and the three VL CDRs ofSEQ ID NO: 1), comprises a VH comprising a sequence at least 95%identical to the VH sequence of SEQ ID NO: 2 and a VL comprising asequence at least 95% identical to the VL sequence of SEQ ID NO: 1, andbinds to human, cynomolgus monkey, rat, and/or mouse CCR2.

In certain embodiments, an antibody or antigen-binding fragment thereofdescribed herein binds to human CCR2, comprises the six CDRs of theantibody listed in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VHCDRs of SEQ ID NO: 2 and the three VL CDRs of SEQ ID NO: 1), comprises aVH comprising a sequence at least 96% identical to the VH sequence ofSEQ ID NO: 2 and a VL comprising a sequence at least 96% identical tothe VL sequence of SEQ ID NO: 1, and binds to human, cynomolgus monkey,rat, and/or mouse CCR2. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human CCR2,comprises the six CDRs of the antibody listed in SEQ ID NO: 1 and SEQ IDNO: 2 (i.e., the three VH CDRs of SEQ ID NO: 2 and the three VL CDRs ofSEQ ID NO: 1, comprises a VH comprising a sequence at least 97%identical to the VH sequence of SEQ ID NO: 2 and a VL comprising asequence at least 97% identical to the VL sequence of SEQ ID NO: 1, andbinds to human, cynomolgus monkey, rat, and/or mouse CCR2. In certainembodiments, an antibody or antigen-binding fragment thereof describedherein binds to human CCR2, comprises the six CDRs of the antibodylisted in SEQ ID NO: 1 and SEQ ID NO: 2 (i.e., the three VH CDRs of SEQID NO: 2 and the three VL CDRs of SEQ ID NO: 1), comprises a VHcomprising a sequence at least 98% identical to the VH sequence of SEQID NO: 2 and a VL comprising a sequence at least 98% identical to the VLsequence of SEQ ID NO: 1, and binds to human, cynomolgus monkey, rat,and/or mouse CCR2. In certain embodiments, an antibody orantigen-binding fragment thereof described herein binds to human CCR2,comprises the six CDRs of the antibody listed in SEQ ID NO: 1 and SEQ IDNO: 2 (i.e., the three VH CDRs of SEQ ID NO: 2 and the three VL CDRs ofSEQ ID NO: 1), comprises a VH comprising a sequence at least 99%identical to the VH sequence of SEQ ID NO: 2 and a VL comprising asequence at least 99% identical to the VL sequence of SEQ ID NO: 1, andbinds to human, cynomolgus monkey, rat, and/or mouse CCR2.

In certain embodiments, a compound of formula (I) is combined with anantibody, antibody fragment or antigen-binding fragment of an antibodythat binds PD-1 and/or an antibody, antibody fragment and/orantigen-binding fragment of an antibody that binds PD-L1. PD-1 is animmune checkpoint protein expressed on activated T cells, B cells, andmonocytes that, upon binding of its ligand PD-L1, regulates the immunesystem, e.g., by promoting apoptosis of antigen-specific T cells andreducing apoptosis of regulatory T cells. PD-L1 can be expressed bytumors to help tumors evade detection and elimination by the immunesystem. Antagonistic inhibition of the PD-1/PD-L1 interactionadvantageously increases T cell activation and enhances recognition andelimination of tumor cells by the immune system. In certain embodiments,the anti-PD-1 antibody is selected from the group consisting ofPembrolizumab, Nivolumab, Cemiplimab, Pimivalimab, Spartalizumab,Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, Dostarlimab,Ezabenlimab, INCMGA0012, AMP-224, AMP-514, SYM-021, LZM-009, CS-1003,SYN-125, GNR-051, MW-11, TY-101, BAT-1306, F520, Sasanlimab, Penpulimab,Pucotenlimab, CX-188, Zimberelimab, and Tebotelimab, or an antibodywhich can compete with Pembrolizumab, Nivolumab, Cemiplimab,Pimivalimab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab,Toripalimab, Dostarlimab, Ezabenlimab, INCMGA0012, AMP-224, AMP-514,SYM-021, LZM-009, CS-1003, SYN-125, GNR-051, MW-11, TY-101, BAT-1306,F520, Sasanlimab, Penpulimab, Pucotenlimab, CX-188, Zimberelimab, orTebotelimab for binding to human PD-1 or a portion of PD-1.

In some embodiments, the anti-PD-1 antibody is Pembrolizumab.

In certain embodiments, the anti-PD-L1 antibody is selected from thegroup consisting Atezolizumab, Avelumab, Durvalumab, Cosibelimab,MSB-2311, ZKAB-001, FAZ-053, MDX-1105, CBT-502, IMC-001, RC-98, KL-A167,GR-1405, Lodapolimab, Sugemalimab, Envafolimab, Opucolimab, andGarivulimab, or an antibody which can compete with Atezolizumab,Avelumab, Durvalumab, Cosibelimab, MSB-2311, ZKAB-001, FAZ-053,MDX-1105, CBT-502, IMC-001, RC-98, KL-A167, GR-1405, Lodapolimab,Sugemalimab, Envafolimab, Opucolimab, or Garivulimab for binding tohuman PD-L1 or a portion of PD-L1.

In some embodiments, the anti-PD-L1 antibody is Atezolizumab.

Further anti-PD-1 antibodies useful in combination with a compound offormula (I) are NAT105 (abcam ab5287); CAL20 (abcam ab237728); EPR20665(abcam ab214421); NAT105-chimeric (abcam ab216352); EPR4877(2) (abcamab137132); EP23119-111 (abcam ab 243644); SP269 (abeam ab227681);PDCD1/1410R (abeam ab218475); EH12.22H7 (abeam ab 223562); PDCD1/922(abeam ab216037); J43 (abeam ab95789); J43.1 (abeam ab 218768); SPM597(abeam ab218474); J116 (abeam ab171267); RMP1-14 (abeam ab171265);EPR18017-203 (abeam ab242810); EPR18017-253 (abeam ab242562);EPR22234-127 (abeam ab259656); EPR22234-42 (abeam ab259655); MAB10861(R&D Systems); MAB10864 (R&D Systems); MAB1086 (R&D Systems); MAB10863(R&D Systems); MAB8578 (R&D Systems); MAB77381 (R&D Systems); MAB7738(R&D Systems); MAB10866 (R&D Systems); MAB10865 (R&D Systems); MAB10867(R&D Systems); SJ01-91 (HUABIO); 1F2 (HUABIO); 3A11 PD-1 blocking Ab(HUABIO); J43 (MyBioSource); RMP1-30 (MyBioSource); 8A1 (BIOSS Inc.);BSR1 (Abeomics); PDCD1/922 (Abeomics); PD1.3.1.3 (Miltenyi Biotec);abx174170 (Abbexa); PDCD1 (Fitzgerald Industries Intl.); J116 (UnitedStates Biological); BSR1 (Nordic BioSite); PDCD1 (BosterBio); 10B3(ProSci Inc.); 4C7 (ProSci Inc.); mhT28 blocking (Sino Biological Inc.);HF06 neutralizing (Sino Biological Inc.); or TK12-02 (CreativeDiagnostics) or an antibody which can compete with anyone of theforegoing antibodies for binding to PD-1 or a portion of PD-1.

Further anti-PD-L1 antibodies useful in combination with a compound offormula (I) are 28-8 (abeam ab205921); EPR19759 (abeam ab213524); CAL10(abeam ab237726); 73-10 (abeam ab228415); EPR20529 (abeam ab213480);SP142 (abeam ab228462); BLR020E (abeam ab243877); RM1012 (abeamab282458); EPR23546-160 (abeam ab252436); ABM4E54 (abeam ab210931);PDL1/2744 (abeam ab269674); MIH5 (abeam ab269253); 29E.2A3 (abeamab259283); MIH6 (abeam ab80276); BMS-5-28 (abeam ab278010); EPR23939-25(abeam ab278009); MAB1561 (R&D Systems); MAB90871 (R&D Systems); MAB1562(R&D Systems); MAB90783 (R&D Systems); MAB10348 (R&D Systems); MAB1561R(R&D Systems); MAB9078 (R&D Systems); MAB10355 (R&D Systems); MIH1(Invitrogen); MIH5 (Invitrogen); RM320 (Invitrogen); JJ08-95(Invitrogen); 485 (Invitrogen); MA5-37856 (Invitrogen); 10D4(Invitrogen); 15 (Invitrogen); 1-111A (Invitrogen); 2B11D11(Proteintech); OTI2C7 (OriGene); UMAB228 (OriGene); OR-5H8 (OriGene);OTI9E12 (OriGene); UMAB229 (OriGene); OTIliG4 (OriGene); OTI2C11(OriGene); OTI14H4 (OriGene); OTI7D4 (OriGene); OTI9E1 (OriGene);OTI11G4 (OriGene); OTI2F5 (OriGene); OTI9A5 (OriGene); OTI3F5 (OriGene);OTI4G4 (OriGene); OTI9E5 (OriGene); OTI13G7 (OriGene); OTI9E10(OriGene); OTI20G10 (OriGene); OR-5E3 (OriGene); OTI4D4 (OriGene);OTI13D11 (OriGene); OTI8C8 (OriGene); OTI16H9 (OriGene); OTI12G7(OriGene); OTI1B12 (OriGene); OTI2E3 (OriGene); OTI2B12 (OriGene);OR-5E4 (OriGene); BLR020E (Bethyl Laboratories); 3F2 (Abnova); 3D2(Abnova); 2E6 (Abnova); 2E11 (Abnova); 1H3 (Abnova); 2C4 (Abnova); Ac10(Abnova); 3C10 (Abnova); or 4C11 (Abnova) or an antibody which cancompete with any of the foregoing antibodies for binding to PD-L1 or aportion of PD-L1.

The term “antibody,” as used herein, also refers to a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

The term “single domain antibody,” also known as a nanobody, is anantibody fragment consisting of a single monomeric variable antibodydomain with a molecular weight of from about 12 kDa to about 15 kDa.Single body antibodies can be based on heavy chain variable domains orlight chains. Examples of single domain antibodies include, but are notlimited to, V_(H)H fragments and V_(NAR) fragments. See, for example,Harmsen M. M. et al. Applied Microbiology and Biotechnology 77 (1):13-22.

“Antibody fragments” comprise a portion of an intact antibody, generallythe antigen binding or variable region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′).sub.2, and Fv fragments; diabodies;linear antibodies; fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CDR (complementary determiningregion), and epitope-binding fragments of any of the above whichimmunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

An “intact antibody” is one which comprises an antigen-binding variableregion as well as a light chain constant domain (CL) and heavy chainconstant domains, CH1, CH2 and CH3. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present disclosure may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597; for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc.) and human constant region sequences.

Various methods have been employed to produce monoclonal antibodies(MAbs). Hybridoma technology, which refers to a cloned cell line thatproduces a single type of antibody, uses the cells of various species,including mice (murine), hamsters, rats, and humans. Another method toprepare MAbs uses genetic engineering including recombinant DNAtechniques. Monoclonal antibodies made from these techniques include,among others, chimeric antibodies and humanized antibodies. A chimericantibody combines DNA encoding regions from more than one type ofspecies. For example, a chimeric antibody may derive the variable regionfrom a mouse and the constant region from a human. A humanized antibodycomes predominantly from a human, even though it contains nonhumanportions. Like a chimeric antibody, a humanized antibody may contain acompletely human constant region. But unlike a chimeric antibody, thevariable region may be partially derived from a human. The nonhuman,synthetic portions of a humanized antibody often come from CDRs inmurine antibodies. In any event, these regions are crucial to allow theantibody to recognize and bind to a specific antigen. While useful fordiagnostics and short-term therapies, murine antibodies cannot beadministered to people long-term without increasing the risk of adeleterious immunogenic response. This response, called Human Anti-MouseAntibody (HAMA), occurs when a human immune system recognizes the murineantibody as foreign and attacks it. A HAMA response can cause toxicshock or even death.

Chimeric and humanized antibodies reduce the likelihood of a HAMAresponse by minimizing the nonhuman portions of administered antibodies.Furthermore, chimeric and humanized antibodies can have the additionalbenefit of activating secondary human immune responses, such as antibodydependent cellular cytotoxicity.

The intact antibody may have one or more “effector functions” whichrefer to those biological activities attributable to the Fc region (anative sequence Fc region or amino acid sequence variant Fc region) ofan antibody. Examples of antibody effector functions include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called .alpha., .delta., .epsilon., .gamma., and .mu., respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

Useful non-immunoreactive protein, polypeptide, or peptide antibodiesinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factors (“TGF”), such asTGF-.alpha. and TGF-.beta., vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, lectins and apoprotein from lowdensity lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Various procedureswell known in the art may be used for the production of polyclonalantibodies to an antigen-of-interest. For example, for the production ofpolyclonal antibodies, various host animals can be immunized byinjection with an antigen of interest or derivative thereof, includingbut not limited to rabbits, mice, rats, and guinea pigs. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and including but not limited to Freund's (completeand incomplete) adjuvant, mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants arealso well known in the art.

Useful monoclonal antibodies are homogeneous populations of antibodiesto a particular antigenic determinant (e.g., a cancer cell antigen, aviral antigen, a microbial antigen, a protein, a peptide, acarbohydrate, a chemical, nucleic acid, or fragments thereof). Amonoclonal antibody (mAb) to an antigen-of-interest can be prepared byusing any technique known in the art which provides for the productionof antibody molecules by continuous cell lines in culture. Theseinclude, but are not limited to, the hybridoma technique originallydescribed by Kohler and Milstein (1975, Nature 256, 495-497), the humanB cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),and the EBV-hybridoma technique (Cole et al., 1985, MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, and IgD and any subclass thereof. The hybridoma producing the mAbsused in this disclosure may be cultivated in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4,72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Purification of the correct molecule, which isusually performed using affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659(1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion may bewith an immunoglobulin heavy chain constant domain, comprising at leastpart of the hinge, C.sub.H2, and C.sub.H3 regions. The first heavy-chainconstant region (C.sub.H1) may contain the site necessary for lightchain binding, present in at least one of the fusions. Nucleic acidswith sequences encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

Bispecific antibodies may have a hybrid immunoglobulin heavy chain witha first binding specificity in one arm, and a hybrid immunoglobulinheavy chain-light chain pair (providing a second binding specificity) inthe other arm. This asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation (WO 94/04690; Suresh et al., Methods in Enzymology, 1986,121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carteret al., 1992, Bio/Technology 10:163-167; Carter et al., 1995, J. ofHematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology16:677-681. Using such techniques, bispecific antibodies can be preparedfor conjugation as ADC in the treatment or prevention of disease asdefined herein.

Hybrid or bifunctional antibodies can be derived either biologically,i.e., by cell fusion techniques, or chemically, especially withcross-linking agents or disulfide-bridge forming reagents, and maycomprise whole antibodies or fragments thereof (EP 105360; WO 83/03679;EP 217577).

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to cancer cell antigens,viral antigens, or microbial antigens or other antibodies bound to tumorcells or matrix. In this regard, “functionally active” means that thefragment, derivative or analog is able to elicit anti-anti-idiotypeantibodies that recognize the same antigen that the antibody from whichthe fragment, derivative or analog is derived recognized. Specifically,in an exemplary embodiment the antigenicity of the idiotype of theimmunoglobulin molecule can be enhanced by deletion of framework and CDRsequences that are C-terminal to the CDR sequence that specificallyrecognizes the antigen. To determine which CDR sequences bind theantigen, synthetic peptides containing the CDR sequences can be used inbinding assays with the antigen by any binding assay method known in theart (e.g., the BIA core assay) (See, for e.g., Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md.; Kabat E et al., 1980, J. ofImmunology 125 (3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)2 fragments, which contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain can beproduced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Other useful antibodies are heavy chain and lightchain dimers of antibodies, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., (1989) Nature334:544-54), or any other molecule with the same specificity as theantibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal and humanimmunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397). Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089) Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in WO 87/02671; EP184,187; EP 171496; EP 173494; WO 86/01533; U.S. Pat. No. 4,816,567; EP12023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987,Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985,Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,BioTechniques 4: 214; U.S. Pat. No. 5,225,539; Jones et al., 1986,Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al., 1988, J. Immunol. 141:4053-4060.

Completely human antibodies can be produced using transgenic mice thatare incapable of expressing endogenous immunoglobulin heavy and lightchains genes, but which can express human heavy and light chain genes.The transgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of a polypeptide of the disclosure.Monoclonal antibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806. Other human antibodies canbe obtained commercially from, for example, Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Biotechnology12:899-903). Human antibodies can also be produced using varioustechniques known in the art, including phage display libraries(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991)).

The antibody may be a fusion protein of an antibody, or a functionallyactive fragment thereof, for example in which the antibody is fused viaa covalent bond (e.g., a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, such as at least 10, 20 or 50 amino acid portion of theprotein) that is not the antibody. The antibody or fragment thereof maybe covalently linked to the other protein at the N-terminus of theconstant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation, thederivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

The antibodies in antibody drug conjugates include antibodies havingmodifications (e.g., substitutions, deletions or additions) in aminoacid residues that interact with Fc receptors. In particular, antibodiesinclude antibodies having modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor (see, e.g., WO 97/34631). Antibodies immunospecificfor a cancer cell antigen can be obtained commercially, for example,from Genentech (San Francisco, Calif.) or produced by any method knownto one of skill in the art such as, e.g., chemical synthesis orrecombinant expression techniques. The nucleotide sequence encodingantibodies immunospecific for a cancer cell antigen can be obtained,e.g., from the GenBank database or a database like it, the literaturepublications, or by routine cloning and sequencing.

The antibody of the ADC may be a monoclonal antibody, e.g. a murinemonoclonal antibody, a chimeric antibody, or a humanized antibody. Theantibody may be an antibody fragment, e.g. a Fab fragment.

Known anti-CCR2 antibodies for the treatment or prevention of cancer canbe conjugated as ADCs. Antibodies immunospecific for a cancer cellantigen can be obtained commercially or produced by any method known toone of skill in the art such as, e.g., recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a cancer cell antigen can be obtained, e.g., from the GenBankdatabase or a database like it, the literature publications, or byroutine cloning and sequencing. Examples of antibodies available for thetreatment of cancer include, but are not limited to, STI-B020X(anti-CCR2 monoclonal antibodies, Sorrento Therapeutics), MC-21(anti-CCR2 humanized antibodies, University of Regensburt/MRC; describedin EP Patent No. 2004692 which is incorporated herein by reference),4.40A68G (Pfizer/Amgen; described in U.S. Pat. No. 8,710,191 which isincorporated herein by reference), UniTI-101 (CSF-1R×CCR2 bispecificantibodies, Elstar Therapeutics), and those described in WO97/31949,which is incorporated herein by reference.

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% sequence identity with at least one receptor bindingdomain of a native antibody or with at least one ligand binding domainof a native receptor, and typically, they will be at least about 80%,more typically, at least about 90% homologous by sequence with suchreceptor or ligand binding domains. The amino acid sequence variantspossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence of the native amino acid sequence. Aminoacids are designated by the conventional names, one-letter andthree-letter codes.

“Sequence identity” is defined as the percentage of residues in theamino acid sequence variant that are identical after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Methods and computer programs for thealignment are well known in the art. One such computer program is “Align2,” authored by Genentech, Inc., which was filed with user documentationin the United States Copyright Office, Washington, D.C. 20559, on Dec.10, 1991.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. An exemplary FcR is a nativesequence human FcR. Moreover, a FcR may be one which binds an IgGantibody (a gamma receptor) and includes receptors of the Fc.gamma.RI,Fc.gamma.RII, and Fc.gamma. RIII subclasses, including allelic variantsand alternatively spliced forms of these receptors. Fc.gamma.RIIreceptors include Fc.gamma.RIIA (an “activating receptor”) andFc.gamma.RIIB (an “inhibiting receptor”), which have similar amino acidsequences that differ primarily in the cytoplasmic domains thereof.Activating receptor Fc.gamma.RIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (Seereview M. in Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991);Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab.Clin. Med., 126:330-41 (1995). Other FcRs, including those to beidentified in the future, are encompassed by the term “FcR” herein. Theterm also includes the neonatal receptor, FcRn, which is responsible forthe transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.,117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g., an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods,202:163 (1996), may be performed.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (VH) followedby a number of constant domains. Each light chain has a variable domainat one end (VL) and a constant domain at its other end. The constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain, and the light-chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light chain and heavy chainvariable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a .beta.-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the .beta.-sheet structure. The hypervariable regions in each chain areheld together in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al supra) and/or those residues from a “hypervariableloop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol.,196:901-917). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa and lambda,based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. The Fv polypeptide may further comprise a polypeptidelinker between the VH and VL domains which enables the scFv to form thedesired structure for antigen binding. For a review of scFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).Anti-ErbB2 antibody scFv fragments are described in WO 93/16185; U.S.Pat. Nos. 5,571,894; and 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(VH) connected to a variable light domain (VL) in the same polypeptidechain (VH-VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et al (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Humanization is a method to transfer the murine antigenbinding information to a non-immunogenic human antibody acceptor, andhas resulted in many therapeutically useful drugs. The method ofhumanization generally begins by transferring all six murinecomplementarity determining regions (CDRs) onto a human antibodyframework (Jones et al, (1986) Nature 321:522-525). These CDR-graftedantibodies generally do not retain their original affinity for antigenbinding, and in fact, affinity is often severely impaired. Besides theCDRs, select non-human antibody framework residues must also beincorporated to maintain proper CDR conformation (Chothia et al (1989)Nature 342:877). The transfer of key mouse framework residues to thehuman acceptor in order to support the structural conformation of thegrafted CDRs has been shown to restore antigen binding and affinity(Riechmann et al., (1992) J. Mol. Biol. 224, 487-499; Foote and Winter,(1992) J. Mol. Biol. 224:487-499; Presta et al., (1993) J. Immunol. 151,2623-2632; Werther et al., (1996) J. Immunol. Methods 157:4986-4995; andPresta et al (2001) Thromb. Haemost. 85:379-389). For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a hypervariable region of the recipient are replacedby residues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit or nonhuman primate having thedesired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see U.S. Pat. No. 6,407,213; Joneset al (1986) Nature, 321:522-525; Riechmann et al (1988) Nature332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native or wildtype sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild type, or modified forms ofan antibody. A parent antibody is directed against a target antigen ofinterest. Antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, or more than 99% by weight, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence by use of a gas phase protein sequencer, or (3) to homogeneityby SDS-PAGE under reducing or nonreducing conditions using Coomassieblue or silver stain. Isolated antibody includes the antibody in situwithin recombinant cells since at least one component of the antibody'snatural environment will not be present. Ordinarily, however, isolatedantibody will be prepared by at least one purification step.

An antibody “which binds” a molecular target or an antigen of interestis one capable of binding that antigen with sufficient affinity suchthat the antibody is useful in targeting a cell expressing the antigen.

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder,such as the development or spread of cancer. For purposes of thisdisclosure, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to a coat protein on the surface of phage,e.g., filamentous phage, particles. One utility of phage display lies inthe fact that large libraries of randomized protein variants can berapidly and efficiently sorted for those sequences that bind to a targetmolecule with high affinity. Display of peptide and protein libraries onphage has been used for screening millions of polypeptides for ones withspecific binding properties. Polyvalent phage display methods have beenused for displaying small random peptides and small proteins, typicallythrough fusions to either PIII or PVIII of filamentous phage. Wells andLowman, Curr. Opin. Struct. Biol., 3:355-362 (1992), and referencescited therein. In monovalent phage display, a protein or peptide libraryis fused to a phage coat protein or a portion thereof, and expressed atlow levels in the presence of wild type protein. Avidity effects arereduced relative to polyvalent phage so that sorting is on the basis ofintrinsic ligand affinity, and phagemid vectors are used, which simplifyDNA manipulations. Lowman and Wells, Methods: A companion to Methods inEnzymology, 3:205-0216 (1991). Phage display includes techniques forproducing antibody-like molecules (Janeway, C., Travers, P., Walport,M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, NewYork, p 627-628).

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., ColE1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle. The compounds describedherein can be in the form of pharmaceutically or pharmaceuticallyacceptable salts. In some embodiments, such salts are derived frominorganic or organic acids or bases. For reviews of suitable salts, see,e.g., Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: TheScience and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), LippincottWilliams & Wilkins (2000).

In the present disclosure, group “Ab” (i.e., the antibodies, antibodyfragments, and/or antigen fragments) can be conjugated to more than onedrug-containing moiety. In some embodiments, “Ab” can be conjugated tofrom 1 to 20 drug-containing moieties. In some embodiments, “Ab” can beconjugated to from 1 to 10 drug-containing moieties. In someembodiments, “Ab” can be conjugated to from 1 to 5 drug-containingmoieties. In some embodiments, “Ab” can be conjugated to from 1 or 2drug-containing moieties. In some embodiments, “Ab” can be conjugated toone drug-containing moiety.

In some aspects of the present disclosure, the ADC is combined with anantibody that binds PD-1 and/or an antibody that binds PDL-1.

The compounds described herein can be in the form of pharmaceutically orpharmaceutically acceptable salts. In some embodiments, such salts arederived from inorganic or organic acids or bases. For reviews ofsuitable salts, see, e.g., Berge et al., J. Pharm. Sci., 1977, 66, 1-19and Remington: The Science and Practice of Pharmacy, 20th Ed., A.Gennaro (ed.), Lippincott Williams & Wilkins (2000).

Examples of suitable acid addition salts include acetate, adipate,alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate,citrate, camphorate, camphor sulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, lucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

Examples of suitable base addition salts include ammonium salts; alkalimetal salts, such as sodium and potassium salts; alkaline earth metalsalts, such as calcium and magnesium salts; salts with organic bases,such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts withamino acids such as arginine, lysine, and the like.

For example, Berge lists the following FDA-approved commerciallymarketed salts: anions acetate, besylate (benzenesulfonate), benzoate,bicarbonate, bitartrate, bromide, calcium edetate(ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate,chloride, citrate, dihydrochloride, edetate(ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate),estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate,gluceptate (glucoheptonate), gluconate, glutamate, glycollylarsanilate(glycollamidophenylarsonate), hexylresorcinate, hydrabamine(N,N′-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate),lactate, lactobionate, malate, maleate, mandelate, mesylate(methanesulfonate), methylbromide, methylnitrate, methylsulfate, mucate,napsylate (2-naphthalenesulfonate), nitrate, pamoate (embonate),pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate(8-chlorotheophyllinate) and triethiodide; organic cations benzathine(N,N′-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine; andmetallic cations aluminum, calcium, lithium, magnesium, potassium,sodium and zinc.

Berge additionally lists the following non-FDA-approved commerciallymarketed (outside the United States) salts: anions adipate, alginate,aminosalicylate, anhydromethylenecitrate, arecoline, aspartate,bisulfate, butylbromide, camphorate, digluconate, dihydrobromide,disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide,methylenebis(salicylate), napadisylate (1,5-naphthalenedisulfonate),oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate,propionate, thiocyanate, tosylate and undecanoate; organic cationsbenethamine (N-benzylphenethylamine), clemizole(1-p-chlorobenzyl-2-pyrrolildine-1′-ylmethylbenzimidazole),diethylamine, piperazine and tromethamine(tris(hydroxymethyl)aminomethane); and metallic cations barium andbismuth.

The compounds described herein may also comprise suitable carriers,excipients, and auxiliaries that may differ depending on the mode ofadministration.

In some embodiments, the pharmaceutical compositions can be formulatedas a suitable parenteral dosage form. Said formulations can be preparedby various methods known in the art. The pharmaceutical compositions canbe administered directly into the bloodstream, into muscle, or directlyinto an organ. Suitable means for parenteral administration includeintravenous, intraarterial, intraperitoneal, intrathecal,intraventricular, intraurethral, intrasternal, intracranial,intramuscular, and subcutaneous. Suitable devices for parenteraladministration include needle injectors, needle-free injectors, andinfusion techniques.

Parenteral compositions are typically aqueous solutions which maycontain excipients such as salts, carbohydrates and buffering agents.However, the composition may also be formulated a sterile non-aqueoussolution or as a dried form to be used in conjunction with a suitablevehicle such as sterile pyrogen-free water.

The preparation of parenteral compositions under sterile conditions, forexample, by lyophilization, can be readily accomplished using standardtechniques known well to those of skill in the art.

Compositions for parenteral administration can be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted, and programmedrelease. Thus, the compositions can be formulated as a solid,semi-solid, or thixotropic liquid for administration as an implanteddepot providing modified release of the active agent.

The parenteral formulations can be admixed with other suitablepharmaceutically acceptable excipients used in parenteral dosage formssuch as, but not limited to, preservatives.

In another embodiment, the pharmaceutical compositions can be formulatedas suitable oral dosage forms such as tablets, capsules, powders,pellets, suspensions, solutions, emulsions, and the like. Other suitablecarriers can be present such as disintegrants, diluents, chelatingagents, binders, glidants, lubricants, fillers, bulking agents,anti-adherents, and the like.

Oral dosage formulations may also contain other suitable pharmaceuticalexcipients such as sweeteners, vehicle/wetting agents, coloring agents,flavoring agents, preservatives, viscosity enhancing/thickening agents,and the like.

The dose of the pharmaceutical compositions of the present disclosurecan be tailored to the individual patient.

The term “radiation” refers to photon radiation or particle radiation.In some embodiments, the radiation can be photon radiation (x-rays andgamma rays). In such embodiments, the photons can be generated as a highenergy photon beam from radioactive sources such as cobalt or a linearaccelerator. In some embodiments, the radiation can be particleradiation (such as electrons, protons, neutrons, carbon ions, alphaparticles, and beta particles). Particle radiation can be produced bylinear accelerators. In some embodiments, the radiation can be anelectron beam. In some embodiments, the radiation can be a proton beam.In some embodiments, the radiation can be a neutron beam.

In some embodiments, the radiation can be delivered by external beamradiation. In some embodiments, the external beam radiation can bethree-dimensional conformal radiation therapy (3D-CRT). In someembodiments, the external beam radiation can be intensity modulatedradiation therapy (IMRT). In some embodiments, the external beamradiation can be image-guided radiation therapy (IGRT). In someembodiments, the external beam radiation can be intensity modulatedproton therapy (IMPT). In some embodiments, the external beam radiationcan be stereotactic radiosurgery (SRS). In some embodiments, theexternal beam therapy can be fractionated stereotactic radiotherapy. Insome embodiments, the external beam radiation can be stereotactic bodyradiation therapy (SBRT). Examples of machines that deliver SBRT areGamma Knife®, X-Knife®, CyberKnife®, and Clinac®. In some embodiments,the radiation can be administered using a three dimensional conformal orstereotactic body radiation therapy delivery.

In some embodiments the radiation can be delivered by internal radiationtherapy (brachytherapy). In such embodiments, the internal radiationtherapy can be interstitial radiation, for example, using small pellets,seeds, wires or tubes placed close to the cancer or tumor site. In suchembodiments, the internal radiation therapy can be intracavitaryradiation, for example using a container of radioactive material thatcan be placed in a body cavity.

Method of Use of Compounds and Compositions

Certain compounds described herein are STING agonists and thus areuseful in stimulating an immune response in subjects thereof. Thecompositions can be used in the treatment of cancer.

Compounds of the present disclosure show STING modulating/agonisticactivity. Certain compounds of the present disclosure can be superior interms of efficacy expression, pharmacokinetics (e.g., absorption,distribution, metabolism, excretion), solubility (e.g., watersolubility), interaction with other medicaments (e.g., drug-metabolizingenzyme inhibitory action), safety (e.g., acute toxicity, chronictoxicity, genetic toxicity, reproductive toxicity, cardiotoxicity,carcinogenicity, central toxicity) and/or stability (e.g., chemicalstability, stability to an enzyme), and can be useful as a medicament.

A compound of the present disclosure can be used for increasing STINGactivity in a mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow,sheep, monkey, human).

A compound of the present disclosure can be used as a medicament such asan agent for the prophylaxis or treatment of diseases that can beinfluenced by STING (in the present specification, sometimes to beabbreviated as “STING-related diseases”), for example, cancers e.g.,colorectal cancers (e.g., colorectal cancer, rectal cancer, anus cancer,familial colorectal cancer, hereditary nonpolyposis colorectal cancer,gastrointestinal stromal tumor), lung cancers (e.g., non-small-cell lungcancer, small-cell lung cancer, malignant mesothelioma), mesothelioma,pancreatic cancers (e.g., pancreatic ductal carcinoma, pancreaticendocrine tumor), pharynx cancer, larynx cancer, esophageal cancer,stomach cancers (e.g., papillary adenocarcinoma, mucinousadenocarcinoma, adenosquamous carcinoma), duodenal cancer, smallintestinal cancer, breast cancers (e.g., invasive ductal carcinoma,non-invasive ductal carcinoma, inflammatory breast cancer), ovariancancers (e.g., ovarian epithelial cancer, extragonadal germ cell tumor,ovarian germ cell tumor, ovarian low-malignant potential tumor), testistumor, prostate cancers (e.g., hormone-dependent prostate cancer,non-hormone dependent prostate cancer, castration-resistant prostatecancer), liver cancers (e.g., hepatocellular cancer, primary livercancer, extrahepatic bile duct cancer), thyroid cancers (e.g., medullarythyroid carcinoma), renal cancers (e.g., renal cell cancers (e.g., clearcell renal cell cancer), transitional cell cancer of renal pelvis andureter), uterine cancers (e.g., cervical cancer, uterine body cancer,uterus sarcoma), gestational choriocarcinoma, brain tumors (e.g.,medulloblastoma, glioma, pineal astrocytic tumors, pilocyticastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitaryadenoma), retinoblastoma, skin cancers (e.g., basalioma, malignantmelanoma), sarcomas (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissuesarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer,blood cancers (e.g., multiple myeloma, leukemias (e.g., acutemyelogenous leukemia), malignant lymphoma, Hodgkin's disease, chronicmyeloproliferative disease), cancer of unknown primary; a cancer growthinhibitor; a cancer metastasis inhibitor; an apoptosis promoter; anagent for the treatment of precancerous lesions (e.g., myelodysplasticsyndromes); and the like.

In certain embodiments, a compound of the present disclosure can be usedas a medicament for colorectal cancer, breast cancer, skin cancer,malignant lymphoma or lung cancer.

In certain embodiments, a compound of the present disclosure can be usedconcurrently with an antibody therapy. In some embodiments, the antibodytherapy comprises an anti-PD-1 antibody. In some embodiments, theantibody therapy comprises and anti-PD-L1 antibody.

In certain embodiments, a compound of the present disclosure can be usedconcurrently with an antibody therapy and radiation therapy. In someembodiments, the radiation therapy can be photon radiation therapy. Insome embodiments, the radiation therapy can be particle radiationtherapy.

Furthermore, a compound of the present disclosure can be usedconcurrently with a non-drug therapy. To be precise, a compound of thepresent disclosure or the combination agent of the present disclosurecan be combined with a non-drug therapy such as (1) surgery, (2)hypertensive chemotherapy using angiotensin II etc., (3) gene therapy,(4) thermotherapy, (5) cryotherapy, (6) laser cauterization and (7)radiotherapy.

For example, by using a compound of the present disclosure before orafter the above-mentioned surgery and the like, or before or after acombined treatment of two or three kinds thereof, effects such asprevention of emergence of resistance, prolongation of Disease-FreeSurvival, suppression of cancer metastasis or recurrence, prolongationof life and the like may be afforded.

In some embodiments, the present disclosure relates to a method oftreating cancer in a patient by administering to a patient in need ofsaid treating a combination of a compound of formula (I), orpharmaceutically acceptable salt thereof, and radiation.

In some embodiments, the present disclosure relates to a method oftreating cancer in a patient by administering to a patient in need ofsaid treating a combination of a compound of formula (I), orpharmaceutically acceptable salt thereof, one or more checkpointinhibitors, and radiation. In some embodiments the one or morecheckpoint inhibitors comprises an antibody. In some embodiments, theone or more checkpoint inhibitors comprises an anti-PD-1 antibody, Insome embodiments, the one or more checkpoint inhibitors comprises ananti-PD-L1 antibody.

In some embodiments, the present disclosure relates to the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,in combination with a checkpoint inhibitor and radiation for thetreatment of cancer in a patient.

In some embodiments, the present disclosure relates to a compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof, for use in treating cancer in a patient, wherein thepatient is also being treated with one or more checkpoint inhibitors andradiation. In some embodiments, the disclosure relates to a compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt thereof, for use in treating cancer in a patient, wherein thecompound of formula (I), or the pharmaceutically acceptable saltthereof, is in combination with the one or more checkpoint inhibitorsand radiation. In some embodiments, the compound of formula (I) can beadministered simultaneously or sequentially with the checkpointinhibitor, radiation, and/or combinations thereof. In some embodiments,the present disclosure relates to methods of treating cancer comprisingadministering to a patient in need of such treatment, a therapeuticallyeffective amount of a combination of a compound of formula (I), one ormore checkpoint inhibitors, and radiation.

In some embodiments, the radiation can be administered at least 5 hoursbefore administration of the checkpoint inhibitor and/or the compound offormula (I). In some embodiments, the radiation can be administered atleast 10 hours before administration of the checkpoint inhibitor and/orthe compound of formula (I). In some embodiments, the radiation can beadministered at least 20 hours before administration of the checkpointinhibitor and/or the compound of formula (I). In some embodiments, theradiation can be administered at least 40 hours before administration ofthe checkpoint inhibitor and/or the compound of formula (I). In someembodiments, the radiation can be administered at least 80 hours beforeadministration of the checkpoint inhibitor and/or the compound offormula (I).

In some embodiments, the radiation can be administered on each of days1-5 of each week and repeated for 2 to 8 weeks. In some embodiments, theradiation can be administered on each of days 1-5 of each week andrepeated for 6 to 8 weeks. In some embodiments, the radiation can beadministered on each of days 1-5 of each week and repeated for 2 weeks.In some embodiments, the radiation can be administered on each of days1-5 of each week and repeated for 3 weeks. In some embodiments, theradiation can be administered on each of days 1-5 of each week repeatedand for 4 weeks. In some embodiments, the radiation can be administeredon each of days 1-5 of each week and repeated for 5 weeks. In someembodiments, the radiation can be administered on each of days 1-5 ofeach week and repeated for 6 weeks. In some embodiments, the radiationcan be administered on each of days 1-5 of each week and repeated for 7weeks. In some embodiments, the radiation can be administered on each ofdays 1-5 of each week and repeated for 8 weeks.

In some embodiments, the radiation can be administered on any two ofdays 1-5 of each week and repeated for 5 to 8 weeks. In someembodiments, the radiation can be administered on any two of days 1-5 ofeach week and repeated for 6 to 8 weeks. In some embodiments, theradiation can be administered on any two of days 1-5 of each week andrepeated for 5 weeks. In some embodiments, the radiation can beadministered on any two of days 1-5 of each week and repeated for 6weeks. In some embodiments, the radiation can be administered on any twoof days 1-5 of each week and repeated for 7 weeks. In some embodiments,the radiation can be administered on any two of days 1-5 of each weekand repeated for 8 weeks.

In some embodiments, the checkpoint inhibitor can be administered onceevery twelve weeks, once every four weeks, once every three weeks, onceevery two weeks, once every week, twice a week, three times a week, ordaily. In some embodiments, the checkpoint inhibitor can be administeredonce every two weeks. In some embodiments, the checkpoint inhibitor canbe administered once every three weeks. In some embodiments, thecheckpoint inhibitor can be administered once every four weeks. In someembodiments, the checkpoint inhibitor can be administered once everytwelve weeks.

In certain embodiments, the radiation is administered at least 40 hoursbefore administration of the checkpoint inhibitor and/or the compound offormula (I). In certain embodiments, the radiation is administered atleast 30 hours before administration of the checkpoint inhibitor and/orthe compound of formula (I). In certain embodiments, the radiation isadministered at least 20 hours before administration of the checkpointinhibitor and/or the compound of formula (I). In certain embodiments,the radiation is administered at least 10 hours before administration ofthe checkpoint inhibitor and/or the compound of formula (I). In certainembodiments, the radiation is administered at least 5 hours beforeadministration of the checkpoint inhibitor and/or the compound offormula (I). In certain embodiments, the radiation is administered atleast 1 hour before administration of the checkpoint inhibitor and/orthe compound of formula (I).

In some embodiments, the compound of formula (I) and/or the checkpointinhibitor can be administered to the patient from 1 day to 3 monthsafter the patient received treatment with radiation. In someembodiments, the compound of formula (I) and/or the checkpoint inhibitorcan be administered to the patient from 1 day to 2 months after thepatient received treatment with radiation. In some embodiments, thecompound of formula (I) and/or the checkpoint inhibitor can beadministered to the patient from 1 day to 1 month after the patientreceived treatment with radiation. In some embodiments, the compound offormula (I) and/or the checkpoint inhibitor can be administered to thepatient from 1 day to 15 days after the patient received treatment withradiation. In some embodiments, the compound of formula (I) and/or thecheckpoint inhibitor can be administered to the patient from 1 day to 7days after the patient received treatment with radiation.

In some embodiments, the radiation can be administered at a fractiondose of about 1 Gy to about 100 Gy. In some embodiments, the radiationcan be administered at a fraction dose of about 1 Gy to about 50 Gy. Insome embodiments, the radiation can be administered at a fraction doseof about 1 Gy to about 20 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 5 Gy to about 20 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 6 Gy to about 18 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 8 Gy to about 16 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 5 Gy to about 10 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 10 Gy to about 15 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 15 Gy to about 20 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 8 Gy or about 16 Gy.

In some embodiments, the radiation can be administered at a fractiondose of about 1 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 2 Gy. In some embodiments, theradiation can be administered at a fraction dose of about 3 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 4 Gy. In some embodiments, the radiation can be administered at afraction dose of about 5 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 6 Gy. In some embodiments, theradiation can be administered at a fraction dose of about 7 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 8 Gy. In some embodiments, the radiation can be administered at afraction dose of about 9 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 10 Gy. In some embodiments, theradiation can be administered at a fraction dose of about 11 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 12 Gy. In some embodiments, the radiation can be administered at afraction dose of about 13 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 14 Gy. In some embodiments, theradiation can be administered at a fraction dose of about 15 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 16 Gy. In some embodiments, the radiation can be administered at afraction dose of about 17 Gy. In some embodiments, the radiation can beadministered at a fraction dose of about 18 Gy. In some embodiments, theradiation can be administered at a fraction dose of about 19 Gy. In someembodiments, the radiation can be administered at a fraction dose ofabout 20 Gy

In some embodiments, the radiation can be administered in fractions. Insome embodiments, the radiation can be administered in from 1 to 10fractions. In some embodiments, the radiation can be administered infrom 1 to 5 fractions. In some embodiments, the radiation can beadministered in 1 fraction, or in 2 fractions, or in 3 fractions, or in4 fractions, or in 5 fractions. In some embodiments, the radiation canbe administered in 1 fraction or in 3 fractions.

In some embodiments, the radiation can be administered at a fractiondose of about 1-5 Gy for 1-3 fractions. In some embodiments, theradiation can be administered at a fraction dose of about 5-10 Gy for1-3 fractions. In some embodiments, the radiation can be administered ata fraction dose of about 10-15 Gy for 1-3 fractions. In someembodiments, the radiation can be administered at a fraction dose ofabout 15-20 Gy for 1-3 fractions. In some embodiments, the radiation canbe administered at a fraction dose of about 5-10 Gy for 1-3 fractions or15-20 Gy for 1-3 fractions. In some embodiments, the radiation can beadministered at a fraction dose of about 8 Gy for 1 fraction. In someembodiments, the radiation can be administered at a fraction dose ofabout 8 Gy for 3 fraction. In some embodiments, the radiation can beadministered at a fraction dose of about 16 Gy for 1 fraction. In someembodiments, the radiation can be administered at a fraction dose ofabout 8 Gy for 1 fraction, or about 8 Gy for 3 fractions, or about 16 Gyfor 1 fraction.

In addition, it is possible to combine a treatment with a compound ofthe present disclosure or the combination agent of the presentdisclosure with a supportive therapy: (i) administration of antibiotic(e.g., β-lactam type such as pansporin and the like, macrolide type suchas clarithromycin and the like) for the complication with variousinfectious diseases, (ii) administration of high-calorie transfusion,amino acid preparation or general vitamin preparation for theimprovement of malnutrition, (iii) administration of morphine for painmitigation, (iv) administration of a pharmaceutical agent forameliorating side effects such as nausea, vomiting, anorexia, diarrhea,leucopenia, thrombocytopenia, decreased hemoglobin concentration, hairloss, hepatopathy, renopathy, DIC, fever and the like and (v)administration of a pharmaceutical agent for suppressing multiple drugresistance of cancer and the like.

EXAMPLES Definitions

Ab antibody

ACN acetonitrile

ADA anti-drug antibody

ADC antibody drug conjugate

BLQ below limit of quantitation

C Celsius

CCR2 C—C motif chemokine receptor 2

CR complete response

CD cluster of differentiation

DAR drug antibody ratio

DMA N,N-dimethylacetamide

DMSO dimethylsulfoxide

DTT dithiothreitol

F extinction coefficient

ε 0.1% 0.1% solution extinction coefficient

EC₅₀ half maximum effective concentration

EDTA ethylenediaminetetraacetic acid

h hours

HIC hydrophobic interaction chromatography

hIgG human immunoglobulin G

HPLC high pressure liquid chromatography

IACUC Institutional Animal Care and Use Committee

IFN interferon

IgG immunoglobulin G

IgM immunoglobulin M

IL interleukin

IP interferon gamma-induced protein

LC liquid chromatography

LCMS liquid chromatography mass spectrometry

μM micromolar

MCP monocyte chemoattractant protein

MDSC myeloid derived suppressor cells

mL milliliters

MS mass spectrum

MTD maximum tolerated dose

NA not available

OAc acetate

PBS phosphate buffered saline

PEG polyethyleneglycol

QTOF quadrupole time-of-flight

rt room temperature

SEC size exclusion chromatography

STING stimulator of interferon genes

TCEP (tris(2-carboxyethyl)phosphine)

TNF tumor necrosis factor

TPPTS 3,3′,3″-phosphanetriyltris(benzenesulfonic acid) trisodium salt

Tris tris(hydroxymethyl)aminomethane

UFLC ultra fast liquid chromatograph

UV ultraviolet

Analytical Methods Analytical SEC Conditions:

SEC spectra were recorded on a Hewlett-Packard HP1100 or an Agilent 1100Series LC system with Diode Array Detector using a SEC column (typicallyTosoh Biosep TSK Gel, G3000SWxl; P/N 8541; 250A; 5 um; 7.8 mm×300 mm) at280 nm. Mobile phase was 100 mM sodium phosphate, 300 mM sodiumchloride, pH 6.8, 10% acetonitrile (v/v) or 1×PBS. A typical run isisocratic at a flow rate of 1 mL/min for 20 min.

Analytical HIC Conditions:

HIC spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100Series LC system with Diode Array Detector using a HIC column (typicallyTosoh Butyl-NPR, 4.6×35 mm, 2.5 um, P/N: 14947) at 280 nm. Mobile phaseA was 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7, and Mobilephase B was 75% 25 mM sodium phosphate, pH 7, 25% isopropanol. For atypical 20 min run, a 12 min linear gradient from 95%/5% A/B to 100% Bwould be used between initial and final intervals of isocratic flow.

LC-QTOF Conditions:

LCMS spectra were recorded on an Agilent 1260 Bioinert Series LC systemconnected to an Agilent 6545 QTOF mass spectrometer using a reversephase column heated to 80° C. (typically Agilent, PLRP-S, 5 μm, 1000 Å,2.1 mm×50 mm). Various gradients and run times were selected in order tobest characterize the compounds. Mobile phases were based on ACN/watergradients and contained 0.1% formic acid. One example of a solventgradient that was used was 95% mobile phase A (mobile phase A=99%water+1% ACN+0.1% formic acid) to 100% mobile phase B (mobile phaseB=95% ACN+5% water+0.1% formic acid) with conditions shown in Table 1.

TABLE 1 Time Flow (min) (mL/min) % A % B 0 0.35 82 18 1 0.35 82 18 20.35 70 30 19 0.5 50 50 19.5 0.5 10 90 21 0.5 10 90 21.1 0.5 82 18 220.5 82 18

Samples were either intact or reduced (20 uL of 1-5 mg/mL ADC solutiontreated with 4 uL of 0.5M DTT solution at 37° C. for 30 min). Raw datawas deconvoluted within appropriate mass range using Agilent BioConfirmsoftware to obtain protein molecular weight(s), and the Agilent DARCalculator was used to calculate DAR.

LC/MS/MS Conditions:

LC/MS/MS analysis was performed using Shimadzu UFLC LC-20AD XR binarypump and SIL-30AC MP autosampler system and AB SCIEX Triple Quad 4500ESI Mass spectrometry.

Typically, 5 uL sample aliquots were injected into the LC/MS/MS afterpassing through a Waters Xselect C18 CSH 3.5 u 2.1 mm ID×30 mm column.Mobile phase A contained 0.1% formic acid in water, and mobile phase Bcontained 0.1% formic acid in 5% water with 95% acetonitrile. Total runtime was 3 min at 1.5 mL/min with a linear gradient from 100% A to 100%B over 1.5 min flow rate. Initially, the instrument was running at 100%aqueous mobile phase solvent for 0.5 min, and then it was increased to100% organic solvent in next 1.5 min.

Preparative SEC:

Preparative SEC purification was conducted on a Gilson Preparative HPLCsystem with UV Detector using a SEC column (typically GE Superdex 200Increase 10/300 GL). Mobile phase was 1×PBS (pH 7.4). A typical run wasisocratic at a flow rate of 1 mL/min for 30 min. Fraction collection wastriggered based on UV threshold (at 214 and 280 nm).

ADC Concentration:

ADC concentration was calculated from the UV absorbance at 280 nmmeasured by NanoDrop (2000c; Fisher Scientific) coefficient aftersubtraction of the UV absorbance from the corresponding linker-payloadconstructs.

Table 2 lists linker-payload constructs that were used for ADCpreparations. The compounds contain either Compound No. 14 (described inWO2018/100558A2) or Compound I-5c (described in WO2019/092660) as apayload. The syntheses of the linker-payload constructs were describedin PCT Application PCT/IB2020/054400.

TABLE 2 Linker- Payload Construct Structure C-2

C-3

C-4

C-5

C-8

C-9

C-10

C-13

C-15

C-18

C-20

C-21

C-22

C-30

C-38

C-39

C-41

The anti-human CCR2 monoclonal antibody composed of the humanizedvariable domains of the heavy and the light chains of the 1D9 mousemonoclonal antibody and the constant domains of human IgG1 heavy chainand human kappa light chain (humanized 1D9, also called TAK-202, mayalso be referred to as the hIgG1 isotype hereunder) was generated asdescribed in U.S. Pat. No. 7,473,421 B2. The hIgG4 isotype of humanized1D9 was prepared in a manner similar to the method described inAnticancer Research March-April 2006 vol. 26 no. 2A 1057-1063.

Sequence of humanized 1D9 Heavy chain: (SEQ ID NO: 3)EVQLVESGGG LVKPGGSLRL SCAASGFTFS AYAMNWVRQA PGKGLEWVGRIRTKNNNYAT YYADSVKDRF TISRDDSKNT LYLQMNSLKT EDTAVYYCTTFYGNGVWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYFPEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELAGAPSV FLFPPKPKDTLMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYTLPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK  Light chain:(SEQ ID NO: 4) DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL DSDGKTFLNW FQQRPGQSPRRLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFPYTFGQGTRLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAKVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACEVTHQGLSSPV TKSFNRGEC Sequence of humanized 1D9 hIgG4 isotypeHeavy chain: (SEQ ID NO: 5)EVQLVESGGGLVKPGGSLRLSCAASGFTFSAYAMNWVRQAPGKGLEWVGRIRTKNNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTFYGNGVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Light chain: (SEQ ID NO: 6)DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTFLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 1 Procedure for Preparation of Ab-STING Agonist Conjugates ViaStochastic Cysteine Conjugation

To a solution of an anti-CCR2 antibody (humanized 1D9, 10 mg/mL) in 50mM histidine, 125 mM arginine, and pH 6.1 buffer was added TCEP (1 mMsolution in H₂O, 2-3 equiv.). The reaction mixture was purged with argonand incubated at rt or 37° C. for 1-3 h with gentle shaking. The desiredlinker-payload construct (5 mM solution in DMA, 6-9 equiv.) was thenadded slowly into the above mixture. The reaction was purged with argonand incubated at rt or ° C. for another 1-2 h with gentle shaking. Thereaction mixture was purified following the preparative SEC methoddescribed herein to give the ADC. The ADC concentration, percentageaggregation, and DAR were determined by UV absorbance, analytical SEC,and LC-QTOF respectively, as described in analytical methods.

A schematic of this procedure is shown in FIG. 1

An analogous procedure to described above was used to prepare otherantibody conjugates.

Example 2 Preparation of Additional Ab-STING Agonist Conjugates ViaStochastic Cysteine Conjugation

The antibody drug conjugates listed in Table 3 were prepared asdescribed in Example 1, using the linker-payload constructs and antibodyshown as the starting materials.

TABLE 3 humanized Aggrega- ADC Linker- 1D9 tion Yield product payloadisotype Payload DAR % % ADC-B1 C-3 hIgG4 Compound 3.7 BLQ 75 I-5c ADC-B2C-21 hIgG4 Compound 4.3 BLQ 80 No. 14 ADC-B3 C-20 hIgG4 Compound 2.4 BLQ40 I-5c ADC-B5 C-4 hIgG4 Compound 3.4 BLQ 100 No. 14 ADC-B6 C-5 hIgG4Compound 3.1 BLQ 56 I-5c ADC-B7 C-2 hIgG4 Compound 3.2 BLQ 92 I-5cADC-B8 C-10 hIgG4 Compound 3.9 BLQ 50 I-5c ADC-B13 C-8 hIgG4 Compound3.9 BLQ 44 I-5c ADC-B14 C-18 hIgG1 Compound 4.0 BLQ 74 I-5c ADC-B16 C-41hIgG1 Compound 4.1 BLQ 51 I-5c ADC-B17 C-38 hIgG1 Compound 3.8 BLQ 74No. 14

Example 3 Procedure for Preparation of Ab-STING Agonist Conjugates ViaTransglutaminase Conjugation

Deglycosylation: A solution of anti-CCR2 antibody (humanized 1D9,generated as described in U.S. Pat. No. 7,473,421 B2, 60 mg/mL) in 50 mMhistidine, 125 mM arginine, pH 6.1 buffer was diluted with an equalvolume of pH 7.2 PBS. To the solution was added N-Glycosydase F (NewEngland Biolabs, P0704S, 500,000 units/mL, 300 units per 1 mg ofantibody) and the reaction mixture was heated to 37° C. with gentlemixing overnight. The resulting deglycosylated humanized 1D9 wasbuffer-exchanged with PBS pH 7.2.

Transglutaminase conjugation: To the deglycosylated humanized 1D9solution (10˜20 mg/mL) in PBS prepared above was added a 0.1M DMSOsolution of amine-PEG-azide (40 equivalents) followed bytransglutaminase (ACTIVA™, Ajinomoto, 5˜10 mg per 1 mg of antibody). Thereaction mixture was heated to 37° C. with gentle mixing overnight. Theproduct was purified following the preparative SEC method describedherein to give humanized 1D9-NH-PEG-azide.

Strain-promoted azide-alkyne cycloaddition: To a solution of humanized1D9-NH-PEG-azide conjugate prepared above (2˜15 mg/mL in PBS) was addeda 4˜10 mM DMSO solution of the strained-alkyne containing linker-payloadconstructs (3-5 equivalents in which DMSO <10% of the total solventvolume). The resulting solution was gently stirred at rt overnight. Theproduct was purified following the preparative SEC method describedherein to give the ADC. The ADC concentration, percentage aggregation,and DAR were determined by UV absorbance, analytical SEC, and LC-QTOFrespectively, as described in analytical methods.

A schematic of this procedure is shown in FIG. 2 (RG=N₃). An analogousprocedure to described above was used to prepare other antibodyconjugates.

Example 4 Preparation of Ab-STING Agonist Conjugates ViaTransglutaminase Conjugation

The antibody drug conjugates listed in Table 4 were prepared asdescribed in Example 3, using the starting linker-payload constructsshown as the starting material in the table.

TABLE 4 humanized Aggrega- ADC Linker- 1D9 PEG tion Yield productpayload isotype length Payload DAR % % ADC- C-22 hIgG4  2 Compound 1.8BLQ 70 B4 No. 14 ADC- C-15 hIgG4 35 Compound 2.0 BLQ 42 B9 I-5c ADC-C-15 hIgG4 23 Compound 2.1 BLQ 70 B10 I-5c ADC- C-13 hIgG4 23 Compound2.0 BLQ 80 B11 I-5c ADC- C-9 hIgG4  9 Compound 1.7 BLQ 50 B12 I-5c

Example 5 Procedure for Preparation of Ab-STING Agonist Conjugates ViaTransglutaminase Conjugation

Transglutaminase conjugation (following the procedure described inTumey, L. N. et al. Mol. Pharmaceutics 2019, 16, 6, 2795-2807 aftermodification): To a solution of transglutaminase (ACTIVA™, Ajinomoto, 50mg per 1 mg of antibody) in pH 6.1 phosphate buffer was added thedeglycosylated humanized 1D9 solution (10˜20 mg/mL, prepared followingthe deglycosylation procedure described in Example 3) in PBS, followedby a 30 mM cystamine.2HCl (50 equivalents) solution in pH 6.1 phosphatebuffer. The reaction mixture was heated to 37° C. with gentle mixingovernight. The product was purified using HiTrap Protein A HP column (GEHealthcare, 17-0402-01), by first washing with 20 mM phosphate pH 7.0and then eluting ADCs with 0.1M citric acid pH 4.0. Further purificationon the preparative SEC method described herein to give humanized1D9-NH—(CH₂)₂—S—S—(CH₂)₂—NH₂.

Maleimide addition: To a solution of humanized1D9-NH—(CH₂)₂—S—S—(CH₂)₂—NH₂ conjugate prepared above (2-15 mg/mL in 20mM pH 5 NaOAc buffer) was added a 5 mM TPPTS solution in water (5equivalents) at 0° C. The resulting solution was incubated at 0° C.overnight. After removing small molecule by dialysis the solution wasincubated for another 24 h at 0° C. The desired linker-payload construct(5 mM solution in DMA, 2.05 equiv.) was then added slowly into the abovemixture. The reaction was incubated at 0° C. for 1.5-2 h with gentleshaking. The reaction mixture was purified following the preparative SECmethod described herein to give the ADC. The ADC concentration,percentage aggregation, and DAR were determined by UV absorbance,analytical SEC, and LC-QTOF respectively, as described in analyticalmethods.

A schematic of this procedure is depicted in FIG. 2 (RG=SH). Ananalogous procedure to described above was used to prepare otherantibody conjugates.

Example 6 Preparation of Ab-STING Agonist Conjugates ViaTransglutaminase Conjugation

The antibody drug conjugates listed in Table 5 were prepared asdescribed in Example 5, using the starting linker-payload constructsshown as the starting material in the table.

TABLE 5 humanized ADC Linker- 1D9 Aggregation Yield product payloadisotype Payload DAR % % ADC- C-18 hIgG1 Compound 2.0 BLQ 80 B18 I-5cADC- C-39 hIgG1 Compound 2.0 BLQ 89 B19 I-5c ADC- C-38 hIgG1 Compound1.9 BLQ 80 B20 No. 14

Example 7 Preparation of Ab-STING Agonist Conjugates ViaTransglutaminase Conjugation

To the deglycosylated humanized 1D9 solution (10˜20 mg/mL, preparedfollowing the deglycosylation procedure described in Example 3) in PBSwas added 1M Tris, 5M NaCl, pH 8.0 buffer (10˜20% of the total volume)to adjust pH to 8.0. To the solution was added 10 mM DMSO solution ofprimary amine-containing linker-payload constructs (20 equiv) followedby transglutaminase (ACTIVA™, Ajinomoto, 100-150 mg per 1 mg ofantibody). The reaction mixture was heated to 37° C. with gentle mixingovernight. The product was purified using HiTrap Protein A HP column (GEHealthcare, 17-0402-01), by first washing with 20 mM phosphate pH 7.0and then eluting ADCs with 0.1M citric acid pH 4.0. The product wasfurther purified following the preparative SEC method described hereinto give the ADC. The ADC concentration, percentage aggregation, and DARwere determined by UV absorbance, analytical SEC, and LC-QTOFrespectively, as described in analytical methods.

A schematic of the procedure is depicted in FIG. 3. An analogousprocedure to described above was used to prepare other antibodyconjugates.

Example 8 Preparation of Ab-STING Agonist Conjugates ViaTransglutaminase Conjugation

The antibody drug conjugate listed in Table 6 were prepared as describedin Example 7, using the starting linker-payload constructs shown as thestarting material in the table.

TABLE 6 humanized ADC Linker- 1D9 Aggregation Yield product payloadisotype Payload DAR % % ADC- C-30 hIgG1 Compound 1.6 BLQ 64 B15 I-5c

Example 9 Procedure for Preparation of Mouse Ab-STING Agonist ConjugatesVia Stochastic Cysteine Conjugation

To a solution of an anti-mCCR2 MC-21 antibody (UniversitaetsklinikumRegensburg, Regensburg, Germany; described in Mack, M. et al. J Immunol.2001, 166, 4697-4704 and WO 2007/115713) (mIgG2a with L235A-G237A-E318Amutation in heavy chain) in 25 mM sodium citrate, pH 5.5 buffer (3.4mg/mL) was added 0.5M tris, 25 mM EDTA, pH 8 solution (10% of the totalvolume) and TCEP (10 mM solution in H₂O, 20 equiv.). The reactionmixture was purged with argon and incubated at 37° C. for 1.5 h withgentle shaking. The reaction mixture was purified following thepreparative SEC method described herein. The purified reduced antibodysolution was cooled to 4° C. Dehydroascorbic acid solution in DMSO (2mM, 3 equiv. relative to the reduced antibody) was added and theresulting mixture was stored at 4° C. overnight. The solution was warmedto rt, and then the desired linker-payload construct (5 mM solution inDMA, 7 equiv. relative to the reduced antibody) was added slowly. Thereaction was incubated at rt for another 1.5-2 h with gentle shaking.The reaction mixture was purified following the preparative SEC methoddescribed herein to give the ADC. The ADC concentration, percentageaggregation, and DAR were determined by UV absorbance, analytical SEC,and LC-QTOF respectively, as described in analytical methods.

A schematic of this procedure is depicted in FIG. 1.

Example 10 Preparation of Additional Mouse Ab-STING Agonist ConjugatesVia Stochastic Cysteine Conjugation

The antibody drug conjugates listed in Table 7 were prepared asdescribed in Example 9, using the linker-payload constructs and antibodyshown as the starting materials.

TABLE 7 ADC Linker- Aggregation Yield product payload mAb Payload DAR %% ADC- C-38 MC- Compound 3.6 BLQ 63 B21 21 No. 14

Example 11 Plasma Stability Assay Conditions

Test compounds were spiked into 1 mL of plasma at a concentration of 10μg/mL and then 5 equal volume aliquots were dispensed into 2 mLEppendorf microfuge tubes (labeled 0, 24, 48, 72, and 96 hours). For the0 h timepoint tubes were immediately stored at −80° C. and the remainingtubes were incubated at 37° C. with moderate shaking. Aliquots wereremoved from the incubator at their corresponding time point and storedat −80° C. After all samples have been collected, they were thawed at rtand placed on wet ice. 50 μL of each sample was dispensed in triplicateinto 96-well microtiter plate. Samples were quenched with 200 μL of icecold methanol containing 50 nM of internal standard. Samples werevortexed for 2 min then centrifuged at 3000 rpm for 10 min. 185 μL ofsupernatant was transferred to a clean injection plate then dried downunder N₂ gas at 40° C. Dried sample extracts were reconstituted with 100L of LCMS grade water then vortexed for 1 min in preparation forLC-MS/MS analysis.

Each sample was separated by reverse phase HPLC using a Synergi 2.5μPolar-RP 100A C18 column (2.0 mm×30 mm), (Phenomenex®) at 40° C. using agradient consisting of 0.1% formic acid in water (Solvent A) and 0.1%formic acid in acetonitrile (Solvent B). Analytes were detected bypositive ion spray in multiple-reaction monitoring (MRM) mode using aSCIEX API 4500 QTRAP instrument. Percentage payload loss in human,primate and mouse plasma at various time points is reported in Table 8.

TABLE 8 Payload loss (%) in human Payload loss (%) in cyno Payload loss(%) in plasma plasma mouse plasma ADC 1 d 2 d 3 d 4 d 1 d 2 d 3 d 4 d 1d 2 d 3 d 4 d ADC-B2 NA NA NA NA 4.5 9.9 15.6 21.7 NA NA NA NA ADC-B51.0 1.8 2.8 3.3 0.7 0.9 2.2 1.4 2.2 3.9 6.4 7.6 ADC-B6 0.3 1.0 1.6 2.40.6 1.0 1.8 2.1 1.4 2.0 3.1 3.9 ADC-B7 2.5 6.2 11.0 16.0 4.8 10.2 16.922.6 3.2 6.5 9.6 3.3 ADC-B14 1.8 3.4 4.9 5.9 1.4 3.0 3.9 5.5 4.5 6.816.0 19.4 ADC-B17 0.7 1.1 1.6 2.2 0.6 0.8 1.4 1.7 2.7 4.1 11.1 9.9

Example 12 THP1 Dual Lucia Reporter Gene Assay Conditions

THP1-Dual™ KI-hSTING-R232 cells (InvivoGen #thpd-r232) were derived fromthe human THP-1 monocyte cell line by stable biallelic knockout of theendogenous human HAQ STING gene and knockin of the R²³² variant of humanSTING. These cells also stably express inducible secreted Lucialuciferase reporter gene under the control of an ISG54(interferon-stimulated gene) minimal promoter in conjunction with fiveIFN-stimulated response elements (ISRE). The expression of reporter geneallows the study of the IFN regulatory factor (TRF) pathway by assessingthe activity of Lucia luciferase. In addition to human STING andluciferase, these cells were engineered to stably express human CCR2 toallow the study of target-mediated activation of IRF pathway. The THP-1cells express endogenous human CCR2 at a much lower density compared tothat of the engineered cells to over express human CCR2. Therefore theempty vector cells could still be used as the negative control.

On the day of experiment, the cells were plated to a white, 384-wellplate (Corning 356661) at 15,000 cells/25 μL per well density in growthmedia (RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivatedfetal bovine serum, 100 μg/mL Normocin™ 100 U/mL-100 μg/mL Pen-Strep, 10μg/mL of blasticidin, 100 μg/mL of Zeocin, and 1 μg/mL of Puromycin).The cell plates were dosed with 5 μL of the hCCR2-targeting-ADC samplesor compound samples, and then incubated at 37° C. for 20 hours. At theend of the incubation, 10 μL/well of the QUANTI-Luc™ (InvivoGen#rep-qlc1) were added, and luminescence was measured immediately usingthe LeadSeeker.

For the assay method described above, percent luminescence signalinduction for each test ADC or test compound, at various concentrations,was calculated relative to untreated and control treated samples.Compound concentration versus percent signal induction curves werefitted to generate EC₅₀ values. One skilled in the art will appreciatethat the values generated as EC₅₀ values are subject to experimentalvariation. The observed EC₅₀ and Emax are reported in Table 9. The datain Table 9 clearly indicates that conjugation of either Compound No. 14or Compound I-5c to humanized 1D9 or its IgG₄ isotype dramaticallyincreases in vitro potency in the hCCR2-overexpressing THP1 cell line.

TABLE 9 hCCR2- overexpressing THP1 Vector THP1 EC₅₀ EC₅₀ ADC/Compound nMEmax nM Emax ADC-B1 NA NA >370 0.6 ADC-B2 2.63 75.1 193 61.8 ADC-B3 NANA >160 4.6 ADC-B4 NA NA >214 5.3 ADC-B5 NA NA 98.6 24.5 ADC-B14 0.53102 366 48.5 ADC-B15 302 59.2 >1000 1.28 ADC-B16 2.46 107 >1000 34.5ADC-B17 1.14 97.8 >1000 25.8 ADC-B18 48.2 56.1 >1000 1.52 ADC-B19 3.0978.3 >200 2.19 Compound No. 14 760 97 710 129 Compound I-5c 850 100 790126

Example 13 Pharmacokinetics Evaluation in Mouse

For in vivo evaluation of the ADCs in naïve Balb/C mouse, female Balb/Cmice at 6-8 weeks of age (purchased from Jackson Laboratory) were used.Mice were fed with normal diet and housed in a SPF animal facility inaccordance with the Guide for Care and Use of Laboratory Animals andregulations of the Institutional Animal Care and Use Committee. Animalswere kept at a temperature of 18-26° C., a relative humidity of 50±20%and intermittent light and dark cycles of 12 hours with food and wateravailable ad libitum.

Pharmacokinetics of the ADCs were studied following injection of ADCsinto Balb/C mice. Serum samples were taken at various time points andstored frozen for analysis.

The mouse plasma levels of total antibodies and conjugated payloads weremeasured by a 2-in-1 immunocapture based LC/MS assay on a Shimadzu UHPLCsystem interfaced to a Sciex 6500 QTRAP mass spectrometer. Briefly,mouse plasma samples were incubated with anti-human IgG coated magneticbeads for 45 min at room temperature, then non-specifically boundproteins were removed by washing the magnetic beads with PBST (PBSbuffer at pH 7.4, containing 0.05% tween 20) and PBS bufferconsecutively. After that, both naked antibodies (DAR=0) and ADCs(DAR≥1) were eluted from the magnetic beads into 0.1% trifluoroaceticacid. After neutralizing the eluents and spiking in stable isotopelabeled internal standards, one aliquot of sample was pipetted out anddigested with papain for 1 hour at 37° C. then used for the LC/MSanalysis of conjugated payloads. The remaining samples were subjected totrypsin/lys-C digestion for 1 hour at 70° C. then used for the LC/MSanalysis of total antibodies.

The free payload in the circulation was also measured by LC/MS afterperforming plasma protein precipitation. In short, mouse plasma wasmixed with 8 volumes of methanol containing stable isotope labeledinternal standard, then the supernatants were evaporated to dryness at40° C. under a gentle nitrogen stream. Finally, the residues werereconstituted in LC/MS grade water prior to LC/MS analysis.

The PK profile of ADC-B14, ADC-B15, ADC-B16, ADC-B17, and ADC-B18 issummarized in Table 10. Graphical representation of the plasma PK isshown in FIGS. 4-8.

TABLE 10 AUC (last) ADC (payload dose) Half life (h) (h*nM) ADC-B14Conjugated 41 47600 (0.05 mg/kg) payload Antibody 68 14370 ADC-B15Conjugated 53 37282 (0.05 mg/kg) payload Antibody 73 13434 ADC-B16Conjugated 44 39200 (0.05 mg/kg) payload Antibody 58 12300 ADC-B17Conjugated 36 58200 (0.05 mg/kg) payload Antibody 59 20400 ADC-B18Conjugated 49 65400 (0.05 mg/kg) payload Antibody 60 39300

Example 14 Tolerability Evaluation in Mouse

The tolerability of the ADC was evaluated in naïve C57BL/6 mice. OnStudy Day 0, animals were weighed, and then administered with theindicated amounts of ADC (by payload concentration) intravenously.Animals were then weighed regularly (no more than 3 days between eachmeasurement) for at least 14 days post dosing, and body weight loss wascalculated after each measurement based upon the pre-dosing startingweight. Any animals with greater than 20% body weight loss, or whichwere moribund or otherwise exhibited signs of distress exceeding thehumane endpoints of the study, were removed from the study andeuthanized according to the guidelines within the IACUC protocol. Themaximum tolerated dose (MTD) was calculated as the highest dose (bypayload concentration) at which no animals were found dead or needed tobe removed from the study, either due to body weight loss greater than20% or having otherwise exceeded a humane endpoint. The MTD of ADC-B17was 200 μg/kg (by payload concentration, FIG. 9) and the MTD of ADC-B20was 250 μg/kg (by payload concentration FIG. 10).

Example 15 Antitumor Activity Evaluation in Mouse

Efficacy of ADC-B21 compared with Compound No. 14 was evaluated in anMC38 (murine colon adenocarcinoma) tumor bearing C57BL/6 mouse model.For tumor implantation, 1×10⁶ MC38 cells were subcutaneously injectedinto C57BL/6 mice and mice were subsequently monitored for tumor growth.When tumor volumes reached an average of approximately 100 mm³, animalswere randomized by tumor volume, and dosed intravenously with 100 μL ofeither vehicle, Compound No. 14 at 2000 μg/kg, or ADC-B21 at 50 μg/kg.The first day of dosing was considered Study Day 0. Compound No. 14 andvehicle were dosed again on Study Days 3 and 6, while ADC-B21 wasadministered as a single administration on Study Day 0. Tumor volumesand body weight measurements were taken at least twice a week until theend of study, and animals were removed for body weight loss greater than20% from starting body weight, or tumor volumes exceeding 2000 mm³. ByStudy Day 63, Compound No. 14 treated animals had a total of 1 out of 6complete responses, compared with ADC-B21 treatment, which had a totalof 4 out of 6 complete responses.

The graphical representation of the observed antitumor activity is shownin FIG. 11, demonstrating anti-CCR2 ADC's significantly enhancedefficacy at much lower dose level comparing to its payload alone.

Example 16 Toxicity Pharmacodynamics Evaluation in Non-Human Primate

2 ADC variants were evaluated in toxicity studies in the cynomolgusmonkey.

A single dose study was performed with intravenous administration ofADC-B2 at 0.15, 0.5, 1.5, or 5 mg/kg (2 monkeys/sex/group) (proteindose). Administration of 5 mg/kg ADC-B2 was associated with earlymortality in two animals on Day 2, attributed to pulmonary toxicity thatwas similar to previous studies with unconjugated payload (Compound No.14) (clinical signs of decreased body pale mucous membranes, anddecreased heart sounds, and histologic findings of mild pulmonaryvascular congestion and acute alveolar hemorrhage that correlated tomacroscopic red discoloration, intra-alveolar edema and fibrin,increased alveolar macrophages and neutrophilic infiltrates, and in oneanimal, pleural and pericardial effusion). Other findings unique tothese early mortality animals were present in the bone marrow (decreasedhematopoietic cellularity, single cell necrosis and increasedhistiocytes), liver (multifocal random foci of necrosis), and lymphoidtissues (decreased cellularity and/or necrosis of germinal centers inthe spleen, and tonsil, and single cell necrosis in the thymus). Onclinical pathology and cytokine analysis of one early mortality animalwhere samples were available was evidence of pro-inflammatory/acutephase response, and elevations in IP-10, IL-6, MCP 1, and TNF-α cytokinelevels that were similar to animals surviving to terminal euthasia.Histologic findings in animals surviving to terminal euthanasia werelimited to lymph node increased cellularity (due to increases inlymphocytes and histiocytic cells) at ≥1.5 mg/kg, and lymph nodegerminal center necrosis in one animal at 5 mg/kg. Pharmacologicendpoints added to the study consisted of flow cytometry to evaluatemonocyte populations, and identified dose-dependent decreases inrelative percentages of classical, intermediate, and non-classicalmonocytes, and myeloid derived suppressor cells (MDSC) at Day 1: 6 and24 hours postdose with partial recovery by Day 1: 48 hours postdose.

A repeat dose study was performed with intravenous administration ofADC-B17 scheduled every 2 weeks for a total of 3 doses at 0.3, 1, or 3mg/kg (protein dose) (2 monkeys/sex/group); however, due to the earlydeath of 2 animals from the 3 mg/kg dose group following the second doseon Day 15, the remaining 2 animals in Group 4 received a reduced dosageof 2 mg/kg on Day 29 (third/final dose). Repeat administration of ≥0.3mg/kg ADC-B17 was associated with the development of anti-drugantibodies (ADA) in 10 out of 12 animals at 1 or more time points afterDay 15 (1 to 3 orders of magnitude increase in signal/noise ratio),directed mostly against the immunostimulatory payload of the ADC, withsome ADA also observed at the end of the time course toward the antibodycomponent of the ADC. These ADA were associated with decreases inexposure (Cmax) following the third dose in most ADA-positive animals.ADC-B17-related early mortality was observed at ≥1 mg/kg. One animal at1 mg/kg was euthanized in moribund condition on Day 29, approximately 7hours postdose. At 3 mg/kg, 1 animal was found dead approximately 6hours postdose on Day 15, and 1 animal was euthanized in moribundcondition on Day 15, approximately 7 hours postdose. ADC-B17-relatedclinical signs in these animals preceding death included red skin(face), decreased activity, hunched posture, body weight loss, excessivesalivation, eyes partly closed, sunken eyeballs, increased bodytemperature, heart murmur, and/or elevated heart rate and/or respirationrate. The cause of mortality was attributed to immune-related effectsconsidered likely due to immunogenicity/hypersensitivity reactions,although direct effects of ADC-B17 could not be ruled out. Serumchemistry findings from all 3 early decedents were generally similar toanimals that survived to terminal euthanasia and were consistent withsystemic pro-inflammatory response and muscle and/or hepatocellulardamage. Hematology and coagulation parameters were evaluated in theanimal euthanized moribund on Day 29 but not the animals on Day 15;there were minimal decreases in lymphocytes and eosinophils attributedto stress and no changes in coagulation parameters. The observedimmunophenotyping changes were similar to that of the surviving animals,described below. Most microscopic findings in early decedent animals onDay 15 were similar to but more severe than animals that survived toterminal euthanasia and consisted of minimal hepatocellular necrosis andsystemic findings consistent with immune-mediated effects (immune cellinfiltrates in the liver sinusoids, adrenal gland, lung interstitium andspleen; thrombosis of the capillaries of the lung; necrosis/fibrindeposition in the spleen; myocardial degeneration; and micro-hemorrhagesin the adrenal gland and epicardial fat). Additional findings unique toearly decedents were considered secondary to stress or moribund status(decreased thymus cellularity correlating to decreased thymic weight andpancreas acinar cell degeneration). In the animal euthanized moribund onDay 29, the only finding was minimal adrenal gland hemorrhage.

In animals surviving to terminal euthanasia, clinical pathology findingswere observed at ≥0.3 mg/kg on Day 3 consisting of mild to moderateincreases in 1 or more of: aspartate aminotransferase, alanineaminotransferase, glutamate dehydrogenase, and creatine kinase. Thesefindings were consistent with a muscle and/or hepatocellular origin andlacked clear histologic correlates. Other findings on Day 3 wereconsistent with a systemic proinflammatory response/acute phase response(minimal to mild increased globulin and c reactive protein and minimalto mild decreases in total protein, albumin, and albumin/globulin ratio)that correlated histologically to inflammatory cell infiltrates inmultiple tissues, or dehydration (mildly increased urea, creatinine, andphosphorus) with no histologic correlates. Each of these changespartially to completely recovered by Day 30. Additional serum chemistrychanges on Days 14 and/or 30 in males only consisted of mild increasesin globulins, and mildly elevated total bilirubin, both of which wereconsistent with an ongoing acute phase inflammatory response. Hematologyand coagulation findings in individual animals at ≥0.3 mg/kg at terminaleuthanasia on Day 30 consisting of mild increases in white blood cellcounts, neutrophil counts, fibrinogen, and activated partialthromboplastin time, and mild decreases in red blood cell count,hemoglobin, hematocrit. These findings were consistent with a systemicproinflammatory response/acute phase response.

Changes on monocytes and MDSCs in plasma samples were evaluated using aflow cytometry panel designed to evaluate monocyte and MDSC counts, aswell as the CCR2, CD80, and CD86 expression on the monocytes. Findingsfrom this evaluation were consistent with expected pharmacology ≥0.3mg/kg ADC-B17 and consisted of a dose-responsive mild to severe decreasein absolute counts of classical monocytes, non-classical monocytes, andmyeloid derived suppressor cells (MDSC) after each dose at with recoverytoward or above baseline prior to each subsequent dose as measured byflow cytometry. CCR2 expression in the classical monocytes was reducedfollowing dosing, with a recovery to near baseline at all doses prior tosubsequent doses (FIG. 12, top). In addition, the expression of CD80 inboth classical monocytes as well as MDSCs was found to increasefollowing each dose, and then recover to at or below baseline levelsprior to subsequent dosing (FIG. 12, middle and bottom, respectively).

Changes in cytokines were also evaluated in plasma samples, and showedat ≥0.3 mg/kg ADC-B17 consisted of large magnitude dose independentincreases in serum IP-10 and MCP-1 concentrations, potential biomarkersof pharmacology, which peaked at 6 hours postdose and returned ortrended to return to baseline values 24 hours postdose. Additionalelevations in serum IL-1RA, IL-6, TNF-α, and IFN-γ were observed thatpeaked at 6 hours postdose and returned or trended to return to baselinevalues 24 hours postdose or prior to the following dose (FIG. 13).

Histologic findings in animals at terminal euthanasia consisted ofmultifocal hepatocellular necrosis without clinical pathology correlatesat ≥0.3 mg/kg. At ≥1 mg/kg there was minimal to mild decreasedcellularity of both erythroid and myeloid precursors in the bone marrow(correlating to hematology findings of mildly decreased red blood cellsand, in 1 animal, markedly decreased lymphocytes), mixed cellinfiltrates sporadically observed within the adrenal gland and liversinusoids, increased cellularity (mixed cells) of the splenic red pulpwhich correlated to mildly increased spleen weight, and minimal focalhemorrhage in the duidenum or heart. These organs with inflammatory cellinfiltrates/hemorrhage were considered likely part of a systemicproinflammatory response and were not considered direct target organtoxicities. Immunohistochemistry for human IgG, monkey IgG and IgM, C3,and/or C9 was performed in order to determine whether immune complexformation and tissue deposition was present in areas of immune cellinfiltrates and/or tissue damage. No granular deposits indicative ofimmune complex formation were detected.

Example 17 Pharmacokinetics Evaluation in Non-Human Primate

Serum samples were taken from non-human primates dosed with ADC-B17described in example 16 at various time points and stored frozen foranalysis. The monkey plasma levels of total antibodies and conjugatedpayloads were measured by a 2-in-1 immunocapture based LC/MS assay on aShimadzu UHPLC system interfaced to a Sciex 6500+ QTRAP massspectrometer. Briefly, monkey plasma samples were incubated withanti-idiotype antibody coated magnetic beads for 60 min at roomtemperature, then non-specifically bound proteins were removed bywashing the magnetic beads three times with PBS buffer. After that, bothnaked antibodies (DAR=0) and ADCs (DAR≥1) were eluted from the magneticbeads into 0.1% trifluoroacetic acid. After neutralizing the eluents andspiking in stable isotope labeled internal standards, one aliquot ofsample was pipetted out and digested with trypsin/lys-C for 1 hour at60° C., then used for the LC/MS analysis of total antibodies. Theremaining samples were subjected to papain digestion for 1 hour at 37°C., then used for the LC/MS analysis of conjugated payloads.

The free payload in the circulation was also measured by LC/MS afterperforming plasma protein precipitation. In short, monkey plasma wasfirst spiked with stable isotope labeled Compound No. 14 followed byprotein precipitation using methanol, then the supernatants wereevaporated to dryness under a gentle nitrogen stream. Finally, theresidues were reconstituted with ammonium acetate solution prior toLC/MS analysis.

The PK profile of ADC-B17 is summarized in Table 11. Graphicalrepresentation of the plasma PK is shown in FIG. 14.

TABLE 11 AUC (last) ADC-B17 protein dose Half life (h) (h*nM) 3 mg/kgConjugated 25 33310 payload Antibody 40 14470 1 mg/kg Conjugated 17 9648payload Antibody 52 4092 0.3 mg/kg   Conjugated 11 2220 payload Antibody18 885

Example 18 (Prophetic) Combination Therapy with PD-1/PD-L1 Antibodies

The tolerability of the ADC in combination with an anti-PD-1 and/oranti-PD-L1 antibody can be evaluated in naïve C57BL/6 mice.

The ADC and anti-PD-1/anti-PD-L1 combinations that can be used are shownin Table 12.

TABLE 12 Linker-Payload Humanized 1D9 ADC product Construct isotypePayload Antibody ADC-B2 C-21 hIgG4 Compound PD-1 No. 14 ADC-B5 C-4 hIgG4Compound PD-1 No. 14 ADC-B17 C-38 hIgG1 Compound PD-1 No. 14 ADC-B4 C-22hIgG4 Compound PD-1 No. 14 ADC-B20 C-38 hIgG1 Compound PD-1 No. 14ADC-B21 C-38 MC-21 Compound PD-1 No. 14 ADC-B2 C-21 hIgG4 Compound PD-LlNo. 14 ADC-B5 C-4 hIgG4 Compound PD-Ll No. 14 ADC-B17 C-38 hIgG1Compound PD-Ll No. 14 ADC-B4 C-22 hIgG4 Compound PD-Ll No. 14 ADC-B20C-38 hIgG1 Compound PD-Ll No. 14 ADC-B21 C-38 MC-21 Compound PD-Ll No.14

For the tolerability studies, the ADC as shown in Table 12 can be dosedat 0.05 mg/kg and the anti-PD-1 and anti-PD-L1 antibodies can be dosedat 0.5, 5, or 50 mg/kg. Since anti-PD-1 antibody Pembrolizumab does notcross-react with rodent PD-1, mice will receive the rat anti-mouse PD-1antibody J43 and the rat anti-mouse PD-L1 antibody MIH5, each at 0.5, 5,and 50 mg/kg.

On Study Day 0, animals can be weighed, and then intravenouslyadministered the indicated amounts of ADC in combination with theindicates amounts of anti-PD-1 and/or anti-PD-L1 antibodies. Animalswill then be weighed regularly (no more than 3 days between eachmeasurement) for at least 14 days post dosing, and body weight loss canbe calculated after each measurement based upon the pre-dosing startingweight. Any animals with greater than 2000 body weight loss, or whichappears moribund or otherwise exhibits signs of distress exceeding thehumane endpoints of the study, can be removed from the study andeuthanized according to the guidelines within the IACUC protocol. Themaximum tolerated dose (MTD) can be calculated as the highest dose (bypayload concentration+PD-1/PD-L1 antibody concentration) at which noanimals can be found dead or needed to be removed from the study, eitherdue to body weight loss greater than 20% or having otherwise exceeded ahumane endpoint. If satisfactory tolerability is achieved with the ADCsand either anti-PD-1 or anti-PD-L1 antibodies, combination therapieswith ADCs and anti-PD-1 and anti-PD-L1 antibodies can be conducted in asimilar way.

Efficacy Study for Combination Therapy in Mice

Efficacy of the ADCs as shown in Table 12 in combination with theanti-PD-1 antibody J43 or the anti-PD-L1 antibody MIH5 can be tested inthe MC38 (murine colon adenocarcinoma) tumor bearing C57BL/6 mousemodel. For tumor implantation, 1×10⁶ MC38 cells can be subcutaneouslyinjected into C57BL/6 mice and mice can be subsequently monitored fortumor growth. When tumor volumes reach an average of approximately 100mm³, animals can be randomized by tumor volume, and dosed intravenouslywith 100 μL of either vehicle, the respective ADC of Table 12 at 50μg/kg and J43 at 0.5, 5, or 50 mg/kg or MIH5 at 0.5, 5, or 50 mg/kg. Thefirst day of dosing can be considered Study Day 0. Tumor volumes andbody weight measurements can be taken at least twice a week until theend of study, and animals can be removed from the study for body weightloss greater than 20% from starting body weight, or tumor volumesexceeding 2000 mm³. The animals can be evaluated at Study Day 63, forcomplete and partial responses. If satisfactory reduction in tumorvolume is not achieved with the combination of ADCs with eitheranti-PD-1 or anti-PD-L1 antibodies, combination therapies with ADCs andanti-PD-1 and anti-PD-L1 antibodies can be conducted in a similar way.

Efficacy Study for Combination Therapy in Non-Human Primates

The ADC in combination with anti-PD-1 antibody Pembrolizumab oranti-PD-L1 antibody Atezolizumab can be administered intravenously tocynomolgus monkeys every 2 weeks (Day 1 to Day 29) for a total of 3doses at 0.3, 0.5, or 1 mg/kg (protein dose) (2 monkeys/sex/group).Pembrolizumab can be dosed at 0.5 or 15 mg/kg and Atezolizumab can bedosed at 0.5 or 15 mg/kg. Animals can be evaluated for hematological andcoagulation parameters, general serum chemistry, and can be assessedhistologically at the end of the study.

Blood samples can be taken from the non-human primates at various timepoints and monkey plasma levels of total antibodies and conjugatedpayloads can be measured by a 2-in-1 immunocapture based LC/MS assay ona Shimadzu UHPLC system interfaced to a Sciex 6500+QTRAP massspectrometer as described above. The free payload in the circulation canalso be measured by LC/MS after performing plasma protein precipitationas described above.

Example 19 (Prophetic) Combination Therapy with Radiation

Tolerability Study

The tolerability of the ADC in combination with an anti-PD-1 and/oranti-PD-L1 antibody and radiation can be evaluated in naïve C57BL/6mice.

The ADC, anti-PD-1 and/or anti-PD-L1 antibodies and radiationcombinations that can be used are shown in Table 13.

TABLE 13 ADC product Antibody Radiation ADC-B2 PD-1 0.5 Gy ADC-B5 PD-10.5 Gy ADC-B17 PD-1 0.5 Gy ADC-B4 PD-1 0.5 Gy ADC-B20 PD-1 0.5 GyADC-B21 PD-1 0.5 Gy ADC-B2 PD-L1 0.5 Gy ADC-B5 PD-L1 0.5 Gy ADC-B17PD-L1 0.5 Gy ADC-B4 PD-L1 0.5 Gy ADC-B20 PD-L1 0.5 Gy ADC-B21 PD-L1 0.5Gy ADC-B2 PD-1 1 Gy ADC-B5 PD-1 1 Gy ADC-B17 PD-1 1 Gy ADC-B4 PD-1 1 GyADC-B20 PD-1 1 Gy ADC-B21 PD-1 1 Gy ADC-B2 PD-L1 1 Gy ADC-B5 PD-L1 1 GyADC-B17 PD-L1 1 Gy ADC-B4 PD-L1 1 Gy ADC-B20 PD-L1 1 Gy ADC-B21 PL-L1 1Gy

For the tolerability studies, the ADC can be dosed at 0.05 mg/kg, theanti-PD-1 and anti-PD-L1 antibodies can be dosed at 0.5, 5, or 50 mg/kgand the radiation can be dosed at 0.5 Gy and 1 Gy.

On Study Day 0, animals can be weighed, the radiation administered about5 h before the intravenously administration of the indicated amounts ofADC in combination with the indicated amounts of anti-PD-1 J43 and/oranti-PD-L1 MIH5 antibodies. Animals will then be weighed regularly (nomore than 3 days between each measurement) for at least 14 days postdosing, and body weight loss can be calculated after each measurementbased upon the pre-dosing starting weight. Any animals with greater than20% body weight loss, or which appears moribund or otherwise exhibitssigns of distress exceeding the humane endpoints of the study, can beremoved from the study and euthanized according to the guidelines withinthe IACUC protocol. The maximum tolerated dose (MTD) can be calculatedas the highest dose of radiation in the combination therapy regimen atwhich no animals can be found dead or needed to be removed from thestudy, either due to body weight loss greater than 20% or havingotherwise exceeded a humane endpoint. If satisfactory tolerability isachieved with the ADCs, anti-PD-1 or anti-PD-L1 antibodies andradiation, combination therapies with ADCs and anti-PD-1 and anti-PD-L1antibodies and radiation can be conducted in a similar way.

Efficacy Study for Combination with Radiation Therapy in Mice

Efficacy of the ADCs in combination with anti-PD-1 and/or anti-PD-L1antibodies and radiation as shown in Table 13 can be tested in the MC38(murine colon adenocarcinoma) tumor bearing C57BL/6 mouse model. Fortumor implantation, 1×10⁶ MC38 cells can be subcutaneously injected intoC57BL/6 mice and mice can be subsequently monitored for tumor growth.When tumor volumes reach an average of approximately 100 mm³, animalscan be randomized by tumor volume, and irradiated with either 0.5 Gy or1 Gy radiation and intravenously dosed with 100 μL of either vehicle,the respective ADC of Table 13 at 50 μg/kg and anti-PD1 antibody J43 at0.5, 5, or 50 mg/kg or anti-PD-L1 antibody MIH5 at 0.5, 5, or 50 mg/kg.The first day of dosing can be considered Study Day 0. Tumor volumes andbody weight measurements can be taken at least twice a week until theend of study, and animals can be removed from the study for body weightloss greater than 20% from starting body weight, or tumor volumesexceeding 2000 mm³. The animals can be evaluated at Study Day 63, forcomplete and partial responses. If satisfactory reduction in tumorvolume is not achieved with the combination of ADCs with radiation andeither anti-PD-1 or anti-PD-L1 antibodies, combination therapies withADCs and radiation and anti-PD-1 and anti-PD-L1 antibodies can beconducted in a similar way.

Efficacy Study for Combination with Radiation Therapy in Non-HumanPrimates

Cynomolgus monkeys can be treated with 0.8 Gy and 1.2 Gy prior toadministration of ADC and anti-PD-1 and/or anti-PD-L1 antibodies. TheADC in combination with anti-PD-1 antibody Pembrolizumab or anti-PD-L1antibody Atezolizumab can be administered following radiation therapyintravenously to cynomolgus monkeys every 2 weeks (Day 1 to Day 29) fora total of 3 doses at 0.3, 0.5, or 1 mg/kg (protein dose) (2monkeys/sex/group). Pembrolizumab can be dosed at 0.5 or 15 mg/kg andAtezolizumab can be dosed at 0.5 or 15 mg/kg. Animals can be evaluatedfor hematological and coagulation parameters, general serum chemistry,and can be assessed histologically at the end of the study.

Blood samples can be taken from the non-human primates at various timepoints and monkey plasma levels of total antibodies and conjugatedpayloads can be measured by a 2-in-1 immunocapture based LC/MS assay ona Shimadzu UHPLC system interfaced to a Sciex 6500+ QTRAP massspectrometer as described above. The free payload in the circulation canalso be measured by LC/MS after performing plasma protein precipitationas described above. Hematological recovery after radiation andextramedullary toxicity can be assessed in the animals.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary embodiments of the present disclosure ascontemplated by the inventor(s), and thus, are not intended to limit thepresent disclosure and the appended claims in any way.

The present disclosure has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the disclosure that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent disclosure. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: a is an integerfrom 1 to 20; Ab is an anti-CCR2 antibody, anti-CCR2 antibody fragment,or an anti-CCR2 antigen-binding fragment; D is a modulator of STINGactivity comprising an amino group on a guanine base, a guanine basederivative, an adenine base, or an adenine base derivative; and L is alinker that, is covalently bonded to Ab; and is also covalently bondedto said amino group on D.
 2. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein D-L is represented bythe formula (Ia):

wherein:

denotes the point of attachment to Ab; b is an integer from 1 to 20; mis 0, 1, 2, 3, or 4; n is 0 or 1; each R¹ is independently selected fromC₁-C₄alkyl, O—C₁-C₄alkyl, and halogen; R² is selected from C₁-C₄alkyland —(CH₂CH₂O)_(s)—CH₃; wherein s is an integer from 1 to 10; R³ andR^(3′) are each independently selected from hydrogen and C₁-C₃alkyl; andL¹ is a cleavable linker fragment.
 3. (canceled)
 4. A compound of claim2, or a pharmaceutically acceptable salt thereof, wherein: m is 0; n is0; and R³ and R^(3′) are each hydrogen.
 5. A compound of claim 2, or apharmaceutically acceptable salt thereof, wherein L¹ is

wherein:

is the point of attachment to the nitrogen atom of formula (Ia);

is the point of attachment to Ab; t is an integer from 1 and 10; W isabsent or a self-immolative group; Z is absent or a peptide of 2 to 5amino acids; U and U′ are independently absent or a spacer; and Q is aheterobifunctional group; provided that W and Z are not both absent. 6.A compound of claim 5, or a pharmaceutically acceptable salt thereof,wherein W is a self-immolative group selected from

wherein:

is the point of attachment to the carbonyl group; and

is the point of attachment to Z.
 7. (canceled)
 8. A compound of claim 5,or a pharmaceutically acceptable salt thereof, wherein W is

wherein:

is the point of attachment to the carbonyl group, and

is the point of attachment to Z.
 9. A compound of claim 5, or apharmaceutically acceptable salt thereof, wherein Z is a peptide capableof being enzymatically cleaved. 10.-11. (canceled)
 12. A compound ofclaim 5, or a pharmaceutically acceptable salt thereof, wherein Z isAla-Val or Val-Ala.
 13. A compound of claim 5, or a pharmaceuticallyacceptable salt thereof, wherein U′ is absent and U is selected from

wherein:

is the point of attachment to Z;

is the point of attachment to Q; p is an integer from 1 to 6; q is aninteger from 1 to 20; X is O or —CH₂—; and each r is independently 0or
 1. 14. A compound of claim 5, or a pharmaceutically acceptable saltthereof, wherein U′ is absent and U is:

wherein:

is the point of attachment to Z;

is the point of attachment to Q; p is an integer from 1 to 6; q is aninteger from 1 to
 20. 15. A compound of claim 5, or a pharmaceuticallyacceptable salt thereof, wherein Q is a heterobifunctional group whichis attached to U′ or, when U′ is absent, is attached to Ab throughchemical or enzyme-mediated conjugation.
 16. A compound of claim 5, or apharmaceutically acceptable salt thereof, wherein Q is selected from

wherein

is the point of attachment to U or, when U is absent, the point ofattachment to Z; and

is the point of attachment to U′, or, when U′ is absent, the point ofattachment to Ab.
 17. A compound of claim 5, or a pharmaceuticallyacceptable salt thereof, wherein Q is:

wherein:

is the point of attachment to U or, when U is absent, the point ofattachment to Z; and

is the point of attachment to U′, or, when U′ is absent, the point ofattachment to Ab.
 18. A compound of claim 5, or a pharmaceuticallyacceptable salt thereof, wherein t is
 1. 19. A compound of claim 2, or apharmaceutically acceptable salt thereof, wherein R² is —CH₃, and R³ andR^(3′) are each hydrogen.
 20. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein a is from 2 to
 6. 21.A compound of claim 2, or a pharmaceutically acceptable salt thereof,wherein b is
 1. 22.-23. (canceled)
 24. A compound of claim 1, wherein Dis a compound of formula (III):

or a pharmaceutically acceptable salt thereof; wherein X¹⁰ is SH or OH;X²⁰ is SH or OH; Y^(c) is O, S, or CH₂; Y^(d) is O, S, or CH₂; B¹⁰⁰ is agroup represented by formula (B¹-A) or formula (B¹-B):

R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently a hydrogen atom or asubstituent; R¹⁰⁰⁰ is hydrogen or a bond to the carbonyl group offormula (I); Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵ and Y¹⁶ are each independently N orCR^(1a), wherein R^(1a) is hydrogen or a substituent; Z¹¹, Z¹², Z¹³,Z¹⁴, Z¹⁵ and Z¹⁶ are each independently N or C; R¹⁰⁵ is a hydrogen atomor a substituent; B²⁰⁰ is a group represented by formula (B²-A) orformula (B²-B):

R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are each independently a hydrogen atom or asubstituent; R^(100′) is hydrogen or a bond to the carbonyl group offormula (I); Y²¹, Y²², Y²³, Y²⁴, Y²⁵ and Y²⁶ are each independently N orCR^(2a), wherein R^(2a) is hydrogen or a substituent; Z²¹, Z²², Z²³,Z²⁴, Z²⁵ and Z²⁶ are each independently N or C; and R²⁰⁵ is a hydrogenatom or a substituent; wherein R¹⁰⁵ and R²⁰⁵ are each independentlyattached to 2- or 3-position of the 5-membered ring they are attached torespectively; provided that: one of B¹⁰⁰ or B²⁰⁰ is attached to ‘L’ informula (I) through the amino group.
 25. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein D is a compound offormula (IIIa):

or a pharmaceutically acceptable salt thereof; wherein B¹⁰⁰ is a grouprepresented by formula (B¹-A) or formula (B¹-B):

R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷ are each independently a hydrogen atom or asubstituent; R¹⁰⁰⁰ is hydrogen or a bond to the carbonyl group offormula (I); Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵ and Y¹⁶ are each independently N orCR^(1a), wherein R^(1a) is hydrogen or a substituent; Z¹¹, Z¹², Z¹³,Z¹⁴, Z¹⁵ and Z¹⁶ are each independently N or C; R¹⁰⁵ is a hydrogen atomor a substituent; B²⁰⁰ is a group represented by formula (B²-A) orformula (B²-B):

R²³, R²⁴, R²⁵, R²⁶ and R²⁷ are each independently a hydrogen atom or asubstituent; R^(100′) is hydrogen or a bond to the carbonyl group offormula (I); Y²¹, Y²², Y²³, Y²⁴, Y²⁵ and Y²⁶ are each independently N orCR^(2a), wherein R^(2a) is hydrogen or a substituent; Z²¹, Z²², Z²³,Z²⁴, Z²⁵ and Z²⁶ are each independently N or C; and R²⁰⁵ is a hydrogenatom or a substituent; wherein R¹⁰⁵ and R²⁰⁵ are each independentlyattached to 2- or 3-position of the 5-membered ring they are attached torespectively; provided that: one of B¹⁰⁰ or B²⁰⁰ is:

wherein: R¹⁸ is hydrogen or C₁₋₆ alkyl; and R¹⁹ is a halogen atom; andthe other is attached to the ‘L’ group in formula (I) through an —NH—group.
 26. A compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein D is a compound of formula of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein: R¹ and R² areeach independently a hydroxy group or a halogen atom; B¹ is:

R¹⁸ is hydrogen or C₁₋₆ alkyl; R¹⁹ is a halogen atom; B² is:

and Q² and Q⁴ are each independently an oxygen atom or a sulfur atom.27. A compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein D is:

or a pharmaceutically acceptable salt thereof, wherein

is the point of attachment to L.
 28. A compound of claim 1, or apharmaceutically acceptable salt thereof, of formula (VI):

wherein a is an integer from 1 to 6
 29. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein Ab is an antibody orfragment thereof that binds human CCR2 or a portion thereof, and iscapable of blocking binding of a chemokine to CCR2 and inhibiting afunction of CCR2.
 30. A compound of claim 29, or a pharmaceuticallyacceptable salt thereof, wherein the antibody is selected from the groupconsisting of monoclonal antibody 1D9 or an antibody which can competewith 1D9 for binding to human CCR2 or a portion of CCR2; MC-21;STI-B020X; UniTI-101; and 4.40A68G.
 31. A compound of claim 30, or apharmaceutically acceptable salt thereof, wherein the antibody ismonoclonal antibody 1D9 or an antibody which can compete with 1D9 forbinding to human CCR2 or a portion of CCR2.
 32. A compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein Ab is a chimericantibody, a humanized antibody, a human antibody, a mouse antibody, arat antibody, a goat antibody, or a rabbit antibody.
 33. A compound ofclaim 1, or a pharmaceutically acceptable salt thereof, wherein Abcomprises a light chain CDR1 comprising amino acids 24-39 of SEQ ID NO:1; a light chain CDR2 comprising amino acids 55-61 of SEQ ID NO: 1; alight chain CDR3 comprising amino acids 94-102 of SEQ ID NO: 1; a heavychain CDR1 comprising amino acids 31-35 of SEQ ID NO:2; a heavy chainCDR2 comprising amino acids 50-68 of SEQ ID NO:2; and a heavy chain CDR3comprising amino acids 101-106 of SEQ ID NO:2.
 34. A compound of claim1, or a pharmaceutically acceptable salt thereof, wherein Ab comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:
 2. 35. A compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein Ab comprises a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:
 1. 36. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein Ab comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises the amino acid sequence of SEQ IDNO:
 2. 37. A compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein Ab comprises a heavy chain variable region and a lightchain variable region, wherein the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:
 1. 38. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein Ab comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO: 2and a light chain variable region, wherein the light chain variableregion comprises the amino acid sequence of SEQ ID NO:
 1. 39. A compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein Abfurther comprises a heavy chain constant region selected from humanimmunoglobulins IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂ heavy chainconstant regions.
 40. A compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein Ab comprises a light chain constantregion selected from the group consisting of human immunoglobulins IgGκand IgGλ light chain constant regions.
 41. A compound of claim 1, or apharmaceutically acceptable salt thereof, wherein Ab binds to the sameepitope as an antibody comprising a variable heavy chain region of SEQID NO: 2 and a variable light chain region of SEQ ID NO:
 1. 42. Acompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein Ab comprises a heavy chain region of SEQ ID NO:
 3. 43. Acompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein Ab comprises a light chain region of SEQ ID NO:
 4. 44. Acompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein Ab comprises a heavy chain region of SEQ ID NO: 3 and a lightchain region of SEQ ID NO:
 4. 45. A pharmaceutical compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable carriers. 46.-49.(canceled)
 50. A method of treating cancer in a subject in need thereof,the method comprising administering to the subject a pharmaceuticallyacceptable amount of a compound of claim
 1. 51. A method for stimulatingan immune response in a subject in need thereof, the method comprisingadministering to the subject a pharmaceutically acceptable amount of acompound of claim
 1. 52.-60. (canceled)